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Microbial Control of Urban Pests — Cockroaches, Ants and Termites

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Urban pests, represented in this chapter by cockroaches, ants and termites, present a unique challenge for microbial control. They are all known to detect and avoid both chemical and microbial pesticides, they have few natural enemies to integrate with microbial pesticides, the relatively constant environment is often inhibitory to infection by fungi, and few naturally occurring pathogens have been identified to develop. Add to this the expectation of householders to have quick, long-term control the pest and the difficulty of obtaining funding for research on the use of pathogens for urban pest control (Oi and Hinkle, 1997), and it becomes obvious why this is an underresearched area.
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Chapter VII-20
Microbial control of urban pests cockroaches,
ants and termites
Richard J. Milner
CSIRO
Division of Entomology
Canberra, ACT 2601, Australia
Roberto M. Pereira
USDA-ARS
Center for Medical
Agricultural and Veterinary Entomology
Gainesville, FL 32608, USA
1 Introduction
Urban pests, represented in this chapter by
cockroaches, ants and termites, present a unique
challenge for microbial control. They are all
known to detect and avoid both chemical and
microbial pesticides, they have few natural
enemies to integrate with microbial pesticides,
the relatively constant environment is often
inhibitory to infection by fungi, and few naturally
occurring pathogens have been identified to
develop. Add to this the expectation of house-
holders to have quick, long-term control the
pest and the difficulty of obtaining funding
for research on the use of pathogens for
urban pest control (Oi and Hinkle, 1997), and
it becomes obvious why this is an under-
researched area. However, there are incentives;
an increasing proportion of householders and
owners of buildings are becoming concerned
about the use of synthetic chemicals because of
allergies, other health concerns and, in the case of
landlords, the threat of legal action. In addition,
buildings offer protection from UV and extremes
of temperature thus enhancing the persistence of
microbial pesticides.
The scale of damage caused by these pests and
the value of the urban pest control market has
led a number of companies to consider possible
investments to develop products for this market.
Unfortunately, data on the efficacy of these
products are rare in refereed journals so most
of the methods described in this chapter have
been developed by University or Government
scientists.
Also needed are studies on the dynamics of
insect pathogens when applied to urban environ-
ments, especially inside buildings. Humidity
and temperature requirements during storage
and after application must be determined for
entomopathogens to be used in urban environ-
ments. Only a few papers report field-testing
of pathogens and the methods used are similar
to those developed for testing chemical pesti-
cides. Pathogens offer the potential advantages of
being more acceptable in a household situation,
recycling within the pest population, more
selective than chemicals and compatible with
predators and parasites. As such they can be key
components of an integrated pest managemen
system.
695
L.A. Lacey and H.K. Kaya (eds.), Field Manual of Techniques in Invertebrate Pathology, 695–711.
© 2007 Springer.
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696 Milner and Pereira
2 Cockroaches
Cockroaches are one of the most familiar
of all domestic pests, and they thrive in
buildings provided there is a source of warmth,
moisture and food. However, their pest status
remains controversial. Cockroach problems have
decreased drastically in recent years due to new
active ingredients, formulations and applications
techniques used in roach control (Robinson,
1999). As a consequence, research on biopes-
ticides for roach control has also received less
attention. Also, despite being capable of trans-
mitting a variety of human pathogens, they
have rarely been convincingly implicated in
outbreaks of disease in the population (Roth
and Willis, 1957; Baumholz et al., 1997).
Rather, their pest status is due to them being
“disagreeable” that “their presence in an urban
situation can be psychologically disturbing and
cause considerable mental distress” (Piper and
Frankie, 1978). They also cause allergies and
this is of increasing concern in developed
countries.
Of the 4,000 or so known species of
cockroaches, only a dozen or so can be
considered as pests. The German cockroach,
Blattella germanica, is by far the most serious
pest and occurs all over the temperate world.
Generally, cockroaches are regarded as less
serious pests in the tropics where the more
open houses and warmer climates result in
cockroaches flying in and out of houses;
consequently, their populations do not build
up to the size that cause such anguish in
the temperate world. Two other species, the
American cockroach, Periplaneta americana,
and the oriental cockroach, Blatta orien-
talis, are also common pests. Other more
minor pests will not be considered in this
chapter.
A Biology of cockroaches
Cockroaches are hemimetabolous and among
the most primitive of insects. B. germanica
is a small cockroach growing up to 10–
15 mm long. It favors temperatures around
30
C, can complete its life-cycle in as little
as 6 weeks, and each female can produce
around 300 eggs. Thus, populations of German
cockroaches can build up to high levels in
a short time. B. orientalis is a larger species
measuring 20–24 mm long. It is more tolerant of
cool temperatures than the German cockroach.
At 28
C, it can complete its life-cycle in less
than 1 year, while at cooler temperatures this
time can be greater than 2 years. A female
can produce over 150 eggs. The American
cockroach is a very large insect measuring 28–
44 mm and is the most serious cockroach pest
of the tropics and subtropics. Warm condi-
tions are needed with the optimum temper-
ature range extending up to 33
C. The life-cycle
can be as short as 1 year. The adults often
live for over a year, and females can produce
15 or more oothecae each containing about
18 eggs.
B Pathogens of cockroaches
Cockroaches are remarkably free of natural
pathogens. The literature on cockroach
pathogens was reviewed by Suiter (1997).
The pathogenicity of many of the described
“pathogens” is unknown and few have been
field-tested. The most promising of these
pathogens are fungi, such as Cordyceps
blattae, which were shown by Roth and
Willis (1960) as being highly pathogenic for
German cockroaches. Various Microsporida
and Haplosporida have also been reported but
as with Cordyceps spp., these pathogens are
difficult to mass produce and use as microbial
insecticides. Lonc et al. (1997) reported that
some strains of Bacillus thuringiensis subsp.
kurstaki caused up to 45% mortality when
fed at high concentrations to cockroaches
(B. orientalis, B. germanica and P. americana).
The only pathogens to have been effectively
field tested are nematodes in the genus Stein-
ernema (Appel et al., 1993), while the fungus,
Metarhizium anisopliae, was available for some
years as BioPath
an EcoScience product sold
for control of cockroaches in the USA (Gunner
et al., 1990), but later withdrawn from the
market. There is also a report of various isolates
of Beauveria bassiana (Zukowski and Bajan,
1996) and Paecilomyces fumosoroseus and other
fungi (Steenberg and Vagn-Jensen, 1998) being
pathogenic to German cockroaches.
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VII-20 Urban pests 697
C Field testing of pathogens as microbial
insecticides of cockroaches
Cockroaches live in environments that are
usually too dry for either nematodes or
fungi to recycle and there are no significant
natural enemies. Therefore, to be commer-
cially successful, a microbial insecticide has to
provide a level of control equivalent, or close
to equivalent, of a chemical. A slow kill may
be acceptable, given the safety advantage of
pathogens.
The two methods of field-testing chemicals
are arena trials and tests under real-life condi-
tions. The former allows reproducible results and
can enable valid comparisons among products.
However, arena trials may give misleadingly
good results as inevitably they are an oversim-
plification of the real world. It is important that
the virulence of the pathogen and the behav-
ioral response of the cockroach to the formu-
lation being used must be determined. Methods
for testing virulence have been generally
covered in Manual of Techniques in Insect
Pathology (Goettel and Inglis, 1997). Testing
for repellency is normally done using “Ebeling
Chambers” (Ebeling et al., 1966). No reports
have been published on the repellency or accep-
tance of the BioPath chambers. For locusts,
it has been shown that oil formulations of
M. flavoviride enhance pathogenicity especially
at low humidities (Bateman et al., 1993). This
led to EcoScience developing a modified BioPath
chamber using an oil carrier (Prior, 1996);
however, this version was never marketed. Inter-
estingly oil-based formulations of chemical pesti-
cides are more repellent than other formulations
(Ali et al., 1992). Thus, while the oil formulation
may enhance the efficacy of the Metarhizium
species, this benefit could be offset by a lack of
acceptance by cockroaches.
Arena trials have been described by
Le Patourel (1996) to assess the efficacy of a
chemical pesticide pyrethroid WP and can be
used for biologicals. The main features of this
type of trial are:
1. Conduct tests under environmental conditions that
are favorable for cockroaches such as a temper-
ature of 28
C, relative humidity at 50–60%, 12:12
light:dark cycle, with food and water provided
ad lib.
2. Use wooden arena boxes, at least 50 ×120 ×
13cm, with the base and one end lined with filter
paper, and the remaining three walls lined with
glass plates. A harborage should be provided and
the food and water placed about 5 cm away from
the wall furthest from the harborage.
3. Introduce at least 50 adult German cockroaches
into each arena and allow 3–4 days for them to
acclimatize before applying treatment.
4. Apply candidate microbial pesticide either by
spraying the plywood panels or by placing
bait stations such BioPath chambers in various
positions within the foraging areas of the arenas
just prior to a dark phase to minimize disturbance.
5. Count the number of insects in the foraging area
at intervals such as every 14 days and record the
mortality.
Ying et al., (1996) tested the EcoScience
BioPath chambers, containing M. anisopliae
conidia, against German cockroaches in an arena
test and reported that with 2 chambers/m
2
,
the LT
50
values were 4.1 and 11.9 days for
male and female cockroaches, respectively. It is
likely that relative humidity of the arena will
affect the efficacy of the mycoinsecticide, and
consequently, a relative humidity much higher
than 50% may be preferred. Since there is
transfer of the fungus from live cockroach to
live cockroach (termed Horizontal Transfer™ by
EcoScience), it may be important to continue the
tests for at least 1 month to show the full effect.
Kaakeh et al. (1996) reported that fungus-killed
cockroaches were not cannibalized suggesting
an avoidance by the healthy cockroaches. The
use of combinations of pathogens with other
products (Pachamuthu and Kamble, 2000; Zurek
et al., 2002; Lopes, 2005) may increase field
efficiency of pathogens.
High levels of control are more difficult
to achieve in real-life situations where there
are a complex series of harborages, variable
abundance of food and potential movement
both within an individual apartment and other
apartments in the same block, usually through
plumbing ducts. Publicly owned apartment
blocks are good sites for testing as they offer
a number of similar apartments, enabling repli-
cation, and cockroaches are often a problem.
Manweiler et al. (1993) tested the efficacy of
Steinernema carpocapsae All strain for control
of German cockroaches in apartments. While
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Book_Lacey&Kaya_1402059310_Proof1_February 10, 2007
698 Milner and Pereira
the nematodes gave comparable control to
the chemical pesticide “Combat” (The Clorox
Company, Pleasanton, CA, USA), both treat-
ments left a significant residual populations and
the authors concluded that improvements were
needed such as the use of attractants in the
nematode stations and improved placement.
The important steps in this type of trial are:
1. Select at least 30 similar apartments and place
into three groups so that the cockroach populations
within each group are as similar as possible.
2. Place 10 commercial sticky traps in specific
locations between a vertical surface and an
appliance. Traps should be left in place for 7
days and then returned to the laboratory for
counting. The population density is assessed using
these traps before treatment as well as at various
intervals up to 12 weeks post-treatment.
3. For the microbial treatments, place 12 stations
per apartment baited with nematodes or another
pathogen with 11 in the kitchen and one in the
bathroom. They should be replaced as required
[Manweiler et al. (1993), replaced the nematode
stations after 4 and 8 weeks]. Chemical insec-
ticide stations should be used as a positive insec-
ticide control and placed in a similar manner in
10 other apartments. These should be replaced in
accordance with the manufacturer’s instructions.
At least six apartments should be used as untreated
controls.
4. Assess the results by calculating the mean
percentage change from the controls at different
time intervals (for example, monthly) for both the
microbial treatments and the chemical treatments.
5. Repeat the experiment at a different time of the
year.
D Conclusions
Despite some limited commercial use in the
USA, pathogens have not generally been shown
to be effective against cockroaches. Both fungi
and nematodes can control German cockroaches
effectively in arena trials; however, under more
realistic conditions it is difficult to achieve the
high level of control demanded by the consumer.
Low humidity is undoubtedly a factor which
reduces the efficacy of fungal pathogens. Conse-
quently, further evaluation of oil-based formula-
tions is warranted.
3 Ants
A Pest species and their importance
Two large groups of pest ants can be recog-
nized in the urban environment: (a) ants which
live inside structures, and (b) ants which are
a problem in urban landscapes, but outside
human structures. Included in the first group
are many ants that invade houses and cause no
structural or other type of damage, but annoy
homeowners. Also included in this group are
ants that can damage the structural integrity of
buildings (carpenter ants, acrobat ants), dissem-
inate disease-causing organisms (pharaoh ants
and others), or consume materials or otherwise
cause financial or aesthetic damage. Included
in the second group are ants that may attack
humans or domestic animals, damage landscape
plants and other materials outside human struc-
tures. These include fire ants which are major
problems in the USA and some other countries
such as Australia (Nattrass and Vanderwoude,
2001). A survey of ant problems in the USA
showed that fire ants and other ants are major
problems in 6% and 13% of the households,
respectively (Whitmore et al., 1992).
Because ants are social insects, special
problems arise in controlling them, as well as in
the evaluation of control levels, especially when
entomopathogens and other biological control
agents are considered (Pereira and Stimac,
1997). Due to a low genetic variability among
individuals in an ant nest, effectiveness of
pathogens can be compromised or enhanced
(Sherman et al., 1988). Nest hygiene, self-
grooming and allogrooming (grooming between
individuals in a social unit) can remove or spread
entomopathogens (Oi and Pereira, 1993) and
chemicals secreted by the ants can be harmful
to the development of microorganisms (Schild-
knecht and Koob, 1971; Jouvenaz et al., 1972;
Storey et al., 1991). However, in the nest,
entomopathogens encounter high host densities,
stable temperatures and humidities and no
harmful radiation.
B Entomopathogens used against ants
In the past, urban ant pests have received
minimum attention from researchers working
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VII-20 Urban pests 699
with microbial control. Relatively few pathogens
have been isolated from or tested against ants.
Among tested microorganisms, most studies
have targeted ant problems in agriculture, such
as the leaf-cutting ants (Kermarrec et al., 1986;
Diehl-Fleig et al., 1993). The ant known in the
USA as the red imported fire ant, Solenopsis
invicta, has been the focus of several attempts
to isolate entomopathogens and use them as
biological control agents (Williams et al., 2003).
Similar effort has been initiated with the ant
Myrmica rubra, a recent invasive ant species
in the USA (Groden et al., 2005). The lack of
recognized specific pathogens of ants prevents
further development of microbial control against
urban ants. Recently, new pathogen isolations
from well-studied ant populations (Pereira et al.,
2002, Pereira, 2004) have demonstrated that
ant pathogens are not as rare as once believed
(Holldobler and Wilson, 1990). Nevertheless,
there is a great need for studies on ant pathogens
and their possible application in urban environ-
ments. Only fungi, nematodes, and microsporidia
(now taxonomically placed with the fungi) have
been seriously tested for use in the control
of ant pests (Drees et al., 1992, Briano and
Williams, 1997, Pereira and Stimac, 1997).
The fungi, M. anisopliae and B. bassiana, and
the steinernematid nematode, S. carpocapsae,
have been the focus of studies on microbial
control of ants. Studies involving the field
prevalence and release of the microsporidia,
Thelohania solenopsae (Briano and Williams,
1997), and more recently Vairimorpha invictae
(Oi et al., 2005) may offer future alternative
for control of fire ants. Efforts with bacteria
have been limited to preliminary pathogenicity
tests with mostly unsuccessful results (Miller and
Brown, 1983; Jouvenaz, 1990). These studies
used bacterial isolates usually obtained from ants
and other insects.
Because several ant species have an efficient
filtering system in the adult stage that
prevents the ingestion of particulate material,
entomopathogens which are acquired per os
are usually effective only against some larval
stages. Relatively little effort has been dedicated
to the isolation and testing of pathogens from
ant larvae. Viruses and bacteria, which are
common pathogens in other members of the
order Hymenoptera, are not well represented
among the pathogens isolated from ant species
(Oi and Pereira, 1993; Pereira and Stimac, 1997).
However, recently a virus has been identified
from fire ant populations (Valles et al., 2004)
and further studies may provide information on
practical uses of this and other viruses against
fire ants and other ant pests. Also, recent studies
(Pereira et al., 2002; Pereira, 2004; Pereira,
unpublished) have shown that several pathogens
that affect ant adults are actually acquired during
the larval stages. This may indicate that the larvae
are the most adequate targets for microbial appli-
cations against ants.
C Application methods
While most microbial control efforts against ants
are aimed at developing biopesticides, classical
biological strategies, with the introduction of
the pathogens and reliance on natural spread
of the disease organism, must be considered as
well. Entomopathogens formulated as biopesti-
cides have to be mass produced and applied in
large amounts, whereas entomopathogens used
in classical biological control do not require
mass production and can be seeded into the
ant population using small quantities of infec-
tious material. In both cases however, there is
an expectation of disease establishment in the
ant population, with eventual elimination of the
pest problem, ideally for several years. With the
classical biological control approach, pest control
is expected to be permanent with maintenance
of the disease organism in the ant population.
Biopesticides are designed to give short-term
solutions to ant problems and control over a
period of years is not usually a goal.
The location of an ant nest, whether indoors
or outdoors, is an important factor in controlling
ant pests with microorganisms. Ants found
indoors may originate from outdoor nests and
control measures applied inside buildings may
not eliminate the problem. Outdoor nests may be
easier to locate, but it is still often difficult to
find ant nests and direct application of control
agents to the ants or their nest may not be
possible. Entomopathogens must be applied so
they will reach the nest, and therefore spread
among the entire ant population. Infection of the
foraging ants alone is unlikely to provide lasting
results since only a small proportion of the ant
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700 Milner and Pereira
population engages in food gathering. Applica-
tions of entomopathogens in baits, which are
carried into the nest by the ant workers, provide
solutions in these cases. When ant nests can be
located, direct application of entomopathogens to
the ants and the nest environment is possible.
Indoor applications of entomopathogens must
be considered in light of the possible exposure
to humans and domestic animals. Although
most entomopathogens are safe, care needs to
be exercised in the application and formu-
lation, for example, to avoid inhalation of fungal
conidia. When applications are made outdoors,
fewer restrictions on application methods and
formulations are necessary but climatic factors
may become more important in determining the
effectiveness of formulations and application
methods.
D Testing entomopathogens for the control
of ants indoors
1 Bait stations
Bait stations containing chemical pesticides have
become very common in the control of indoor
cockroaches, ants and other insects. They consist
of small structures with openings allowing
entrance of the insects and contain an attractive
bait and insecticide. This type of application is
very easy for the homeowner to use and avoids
exposure of humans and nontarget animals to
the active ingredient. When chemical pesticides
are used, the bait material usually contains
a slow-acting toxicant. Because the toxicants
do not kill the insect immediately, ants can
transfer the active ingredient to nest mates, and
greater mortality is obtained. Entomopathogens
are naturally slow-acting, and therefore ideal for
use in bait stations.
Bait stations containing the entomopathogenic
fungus B. bassiana caused higher ant mortality
than some commercial ant baits for control of
several urban ant pests (Stimac and Pereira, 1997,
2001, 2006). The following protocol can be used
for testing B. bassiana against several species
of ant:
1. Coat the walls of small plastic boxes 17 ×12 ×
55cm or 19×14 ×95 cm) with liquid Fluon™
or Teflon™ to prevent ants escaping. These boxes
will serve as the arenas where individual ant
colonies will be kept.
2. Add nest cells (Petri dishes partially filled with
either dental plaster or plaster of Paris, which is
kept damp to maintain humidity, and with entrance
holes on the lids or side walls) to the plastic
boxes to provide shelter for the ants. Nest cells
prepared with 60-mm diameter plastic Petri dishes
will accommodate fire ants and carpenter ants, but
a 35-mm can be used for pharaoh ants and other
small species.
3. Prepare artificial ant colonies by separating small
groups of individuals from larger colonies. The
number of ants in each replicate will depend on
the species and availability of ants. Typically,
0.5 g of fire ant workers is used per colony. For
pharaoh ants, artificial colonies should consist of
approximately 150 workers and 1 queen, and for
carpenter ants 50 workers only. Ant colonies may
be prepared 1–3 days before initiation of experi-
ments to allow insects to adapt to the arena and
nest cells.
4. Prepare the bait as a powder containing 35%
ground roast peanuts, 50% cornstarch, 10%
B. bassiana conidia and 5% drying agent (diatoma-
ceous earth or a synthetic calcium silicate). Ensure
the bait powder is free-flowing by passing it
through a 60-mesh or finer sieve.
5. The bait stations, which may consist of a weigh
dish, weighing paper, bait stations used with
chemical pesticides, or any other similar structure,
are filled with 0.5-2.0 g of the free-flowing bait
powder and added to the plastic boxes.
6. Remove and count the dead ants after 3, 7, 14, 21
and 28 days. Finally, count the ants still surviving
after 28 days.
Kelley-Tunis et al. (1995) conducted experi-
ments with carpenter ants and entomopathogenic
fungi using a bait station as the inoculation
chamber. The chamber was similar to commer-
cially available ant bait stations, and the fungal
preparation was placed within the lid, above
an ant-attractive material. Ants were inocu-
lated when B. bassiana and M. anisopliae
conidia were dislodged from the preparation and
contacted them.
2 Other applications
Besides application from fixed stations, baits and
other formulations containing entomopathogens
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VII-20 Urban pests 701
can also be distributed along areas visited by
ants and other insects inside buildings. Fungal
pathogens, which need to contact the insect
cuticle, are probably better used in powdered
formulations that can readily be transferred onto
the insect’s body. Other pathogens, such as
microsporidia, that need to be ingested by the
host, must be formulated in a substrate that will
be eaten by the target insects.
E Testing entomopathogens for control of ants
outdoors
1 Broadcast applications
Both broadcast applications over large areas
and the placement in piles of baits (10 g per
pile, and 24 kg bait/ha) containing B. bassiana
conidia have been used to control the southern
fire ant Solenopsis xyloni (Pereira and Stimac,
unpublished data). The placement of a bait
into piles provides a concentrated source of
attractive volatiles, but decreases chances of
the bait being found randomly by a foraging
ant. Non-attractive powders containing fungal
conidia have also been used in broadcast appli-
cation. A hydrophobic silica was used as the
carrier for this formulation to prevent adherence
of the material to wet soil. The hydrophobic
nature of the carrier also promoted attachment of
the fungal formulation to the insect cuticle as the
ants moved over the treated area.
Alginate granules containing B. bassiana
mycelium have been used in broadcast applica-
tions against fire ants (Bextine and Thorvilson,
2002). Granules coated with peanut oil as an ant
attractant apparently caused some reduction in
fire ant populations. Fungal infection was also
detected in individual ants collected from the
field. However, results of this research have not
been successful enough to lead to commercial
product development.
2 Individual nest applications
If an ant nest can be found, the application
of entomopathogens in or on the nest, or the
area immediately around the nest, will improve
the chance of contact between the pathogen
and the ant host. The application of granular
bait containing chemical pesticides around nests
is commonly recommended (Collins, 1992).
Powder and granular baits containing B. bassiana
have also been applied to Solenopsis spp. nests
with variable results (Bextine and Thorvilson,
2002, Pereira and Stimac, unpublished). Infected
caterpillars have also been used as baits
to introduce B. bassiana into fire ant nests
(Broome, 1974).
The use of injection devices to introduce
pathogens into ant nests has produced good
results (Oi et al., 1994). The procedures used in
these experiments are as follows:
1. Mark the fire ant nests with flags and map their
location in the study area. Assess the level of
ant activity at each nest using an arbitrary rating
system (see below).
2. Collect samples of ants (at least 100 individuals)
by disturbing the nest and placing a spatula on
top of it. Shake the ants climbing on the spatula
into Fluon
-coated trays and transfer them to
samples vials. In the laboratory, kill the ants by
freezing and use them in infection determination
as described below.
3. Inject 7–50 g of a hydrophobic powder formulation
(90% hydrophobic fumed silica (TS-720, Cabot,
Tuscola, IL, USA) and 10% B. bassiana conidia)
into the fire ant nests. The use of a hydrophobic
formulation prevents the adhesion of the powder
to the wet wall of the nest galleries and ensures a
better distribution of the applied material.
4. For step 3, use an injector consisting of a 2-liter
plastic bottle, into which the powder formulation
is added, and a discharge tube and rod, which
was inserted into the ant nest. Compressed gas is
then used to inject the material into the ant nests
(Figure 1).
5. Deposit the powder formulation deep (10–60 cm)
into the ant nest. Injection of the fungal formu-
lation should be stopped when the formulation is
observed emanating from openings at the surface
of the nest. This guarantees a complete coverage of
the nest galleries with biopesticide. Formulations
containing > 90% of fungal conidia mixed with
diatomaceous earth do not produce good coverage
because they do not disperse well within the ant
nests. Such formulations remain at the injection
site which will then be avoided by the ants. This
avoidance is due to the repellent effect of high
fungal content in formulations.
Drench and injection applications of the
nematode, S. carpocapsae, in water suspensions
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Figure 1. Injector used for application of hydrophobic powder formulation containing Beauveria bassiana conidia into fire
ant nests. The formulation is added to the 2-liter plastic bottle. Compressed air from the tank will enter the bottle and suspend
the formulation in air, which escapes through the tube at the top of the bottle. Some of the compressed air bypasses the
formulation bottle and helps to siphon the formulation/air mixture from the formulation bottle. The formulation is injected
into the fire ant nests through the metallic tube which is inserted into the soil. The formulation escapes the tube through holes
at the tip of the metallic tube
have also been used against fire ants. Drenches
(2–5 ×10
6
nematodes in 3.8 liters of water per
nest) were applied to the top and perimeter of
fire ant mounds or injected (2 ×10
6
nematodes
in 0.95 liter of water per nest) 0.5 m into mounds
(Drees et al., 1992). Significant reduction in
the activity of treated nests was observed with
these treatments, but new satellite mounds were
found associated in 32–44 % of nematode-
treated nests. This, and other applications that
severely disturb the ants in the nests, tend to
cause nest movement, limiting the efficacy of the
entomopathogen.
F Evaluation methods
When ants and other social insects are targeted
for control, the colony, rather than the individual,
should be considered as the unit. Thus, the
objective of control tactics must be the elimi-
nation of the reproductive potential in the nests.
If accomplished, the colony will eventually die.
The elimination of workers, may solve the
immediate pest problem, but will not give long-
term benefit. Quantification of percent mortality
or infection of individuals from a nest may serve
as an indicator of the effectiveness of microbial
control measures, but percent nest mortality is
the true measure of success.
Several indicators have been used in evaluating
the effectiveness of entomopathogenic applica-
tions against urban ants:
1 Activity ratings
Activity ratings are usually based on visual
observation of ant response to some disturbing
stimulus. With fire ant colonies, the top of the
nest is touched and disturbed, or air is blown into
the nest, and the time it takes the ants to respond
is evaluated. Ratings are assigned according to
how the nest performs in relation to a normal
response. Fire ant nests can be rated as active if
> 30 ants respond within 20 seconds disturbance,
and inactive otherwise (Oi et al., 1994). Alter-
natively, a “progressive minimal mound distur-
bance technique” can be used and nests rated as
active when > 50 ants respond to an increasing
level of mound disturbance (Drees et al., 1992).
Depending on level of disturbance necessary to
elicit the response, nests can be categorized into
different levels of activity (e.g., high, medium,
low, inactive). Because insect activity can be
affected by several factors including weather
conditions, these ratings may not reflect the
true population levels, or ability of the ants to
cause damage.
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VII-20 Urban pests 703
2 Trapping and populations indexes
Two types of traps can be used to monitor ant
populations: traps that prevent escape of the ants
(pitfall traps, sticky cards), and traps that allow
ants to leave and recruit more foragers. The
former can be left in place for longer periods of
time, but are usually less specific and many other
insects may be caught. The second trap type is
usually baited with attractive material that will
be foraged by the ants. Because ants are free to
leave, these are usually monitored or collected
within minutes to hours of their deployment.
Depending on how specific the attractant is,
a limited number of species can be expected.
Snap-cap tubes (8 cm ×25 cm diameter, 45-ml
capacity) baited with several attractants including
wieners, dog food, canned tuna fish, vegetable
oil, honey water etc. can be used to monitor ants.
Liquid attractants such as oils and honey water
can be applied to absorbent paper (Kimwipe
®
)
placed into the traps. Attractants are placed
within the tubes which are laid sideways on
the ground to allow free access of ants to the
attractive material. Tubes can be placed next to
ant nest entrances, near foraging trails, randomly
in the field or in a grid pattern (e.g., 10×10m),
depending on objective of study. Traps placed
next to nest entrances or by foraging trails
estimate populations of single nests whereas
traps placed over an area, randomly or in a grid,
estimate areawide populations. Snap-cap tubes
can be left out for 10 to 240 minutes, then
capped to trap ants and recovered. Ants are then
freeze-killed and counted. Studies with fire ants
have shown that an adequate estimate of field
populations can be obtained by leaving baits
consisting of wiener slices in the field for 30 to
60 minutes followed by evaluation of the percent
of baits with ants (Pereira, 2003). If less than
30–40% of the baits are occupied by fire ants,
the population is too low to justify application
of control (Pereira, unpublished). Similar evalu-
ation systems may be used for other ants.
A population index which takes into consid-
eration the estimated size of the ant nest and
the presence or absence of brood, has been used
extensively for monitoring fire ant populations
including those affected by an entomopathogen
(Lofgren and Williams, 1983). Depending on the
estimated number of ants in a nest, population
indexes of 1 to 5 are assigned, and when worker
brood is present, rates are multiplied by 2. In
another evaluation system, nests without brood
are rated between 1 and 5, whereas nests with
brood receive rates of 6–10. Trained individuals
are able to rate nests accurately and consistently,
but this population index requires good famil-
iarity with the ant species. Population indicators,
such as size of nests, damage to structures and
others, may also be used depending on the ant
species. A good knowledge of the ant biology
and behavior is necessary for ratings, which are
good representations of the true population.
3 Percent infection
Whether ants are trapped or collected directed
from nest or environment, they can be used
in estimating the percent infection with fungal
or bacterial pathogens using the following
procedure (Pereira et al., 1993):
1. Kill the ants by freezing. Allow enough time for
complete kill since some ant species are quite
resistant to freezing.
2. Surface-sterilize the ants by quickly dipping them
in 95% ethanol (5–10 seconds).
3. Remove ants from ethanol and allow the ethanol to
evaporate by placing the ants on a paper towel, if
possible under a laminar flow hood or other sterile
work area.
4. Place individual ants into empty wells on 96-well
microtiter plates.
5. Cover each plate with moist paper towels or
Kimwipes™.
6. Place plates in a plastic box with moist paper
towel covering the bottom. Plates can be stacked
in the box but the moist paper covering each plate
should touch the moist box bottom. This allows
the water to be drawn onto the paper to maintain a
moist environment within each well on the plates.
Alternatively, ants can be plated on 2% water agar
plates.
7. Incubate plates at 25
C.
8. Observe ants after 5–10 days and record as infected
those showing signs of pathogen growth.
Microscopic observation of wet mounts or
stained slides are used for observation of proto-
zoans and nematodes. Staining and other relevant
techniques are described in several chapters in
Lacey (1997). Most ant diseases can only be
detected in killed or dissected insects. However,
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704 Milner and Pereira
fire ants infected with the protozoan-caused
yellow head disease (Pereira et al., 2002), with
the fungus Myrmicinosporidium durum (Pereira,
2004), or with a yeast infection (Pereira, unpub-
lished) can be diagnosed without being killed.
Like these, other ant pathogens may be detectable
in live insects.
Ants trapped or collected as they respond
to a disturbing or attracting stimulus are not
random samples of the ant population. Ants with
advanced stages of a disease will likely not
be present in these samples. Therefore, these
techniques tend to underestimate the prevalence
of disease in the ant population. Because of the
social behavior of the ants, and behavior modifi-
cations caused by entomopathogenic infections
(Oi and Pereira, 1993), other sample collection
methods may not guarantee a random sample
of the population either. Trapped ants usually
provide a good indication of the prevalence of an
entomopathogen in the ant population. However,
depending on the trapping method and the ant
behavior when affected by the entomopathogen,
the true infection level may be quite different
from the observed one.
4 Pathogen persistence in the environment
Plating of soil and other habitat materials
onto selective medium [e.g., for B. bassiana,
Beilhartz et al. (1982)] can be used to monitor
levels of some of the entomopathogens in the
ant environment (Oi et al., 1994). Obligate
parasites will not be detected using media.
Galleria mellonella (Zimmermann, 1986) or
other suitable hosts can be used as pathogen
bait for detection of fungi, nematodes and
possible bacteria. Several methods described in
Lacey (1997) can be used for detection of the
different pathogens.
4 Termites
Termites belong to the order Isoptera (meaning
equal wings) and some 2,500 species are
recognized today. Although they are essentially
tropical insects, they occur as far north as
southern Canada and as far south as northern
Argentina. Termites are thought to be closely
related to cockroaches and to have evolved
some 200 million years ago (Robinson, 1996).
They are pests of buildings, forestry and crops
over all of Africa, as well as much of Asia,
Australia, South America, North America and
southern Europe. It has been estimated that the
4 or 5 major termite pest species in the USA
are responsible for over $1 billion damage to
buildings and agriculture each year (Robinson,
1996). Termites are undoubtedly the most serious
pest of buildings and human-made structures
world-wide.
A Biology of termites
Termites are social insects living in colonies
comprising of a king and queen together with
numerous workers and soldiers. Their life cycle
is hemimetabolous with the queen laying eggs
that hatch into larvae which can develop into
workers, soldiers or new reproductives. There are
many variations on this simple pattern; colonies
are always long-lived and primary queens often
live for over 20 years and in some cases up
to 50 years. Termites may be divided into
“lower” and “higher” groups. Lower termites
such as Mastotermitidae and Rhinotermitidae
have cellulose-digesting flagellates in their guts,
whereas the higher termites, or Termitidae,
generally do not require specialized protozoans
and have a more complex social organization.
Some 80% of termite species belong to the higher
termites and many cultivate fungi for food within
the nest.
In the USA, the major urban pests are Retic-
ulotermes flavipes and Coptotermes formosanus.
Both species normally form large, diffuse,
subterranean colonies comprising many millions
of individuals and foraging over an area of
1000–2000 m. C. formosanus may also form
nests in trees (Henderson and Forschler, 1997).
Thus, a single colony is often responsible for
damaging several houses and foraging distances
of over 50 m are commonly recorded. C.
formosanus is thought to have been accidentally
introduced into the USA from Asia, possibly
China, while Reticulotermes spp. are becoming
increasingly serious pests in Europe especially in
France.
In Australia, two other Coptotermes spp.,
C. frenchi and C. acinaciformis, are the main
urban pests and both species form discrete nests
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VII-20 Urban pests 705
of many millions of individuals often in trees.
As with C. formosanus and R. flavipes, foraging
distances can be 50 m or more. Mound builders
such as Nasutitermes exitiosus are less serious
pests. In other parts of the world, urban pests may
be subterranean, mound builders or so-called dry-
wood termites. Dry-wood termites are able to
form nests away from the soil and so may even
form nests high up in roof structures. These
colonies can be small and numerous.
B Pathogens of termites
Fungi, such as M. anisopliae and B. bassiana,
can be easily isolated from termites and from
termite derived substances such as mound
material (Zoberi and Grace, 1990; Zoberi,
1995). However, a large detailed survey in
Australia concluded that M. anisopliae was not
a significant natural pathogen (Milner et al.,
1998a). Colonies of termites must eventually
decline and die; however, no pathogens have
yet been shown to be the cause of colony
mortality under natural conditions. Hanel (1982)
listed a number of fungal pathogens recorded
from termites and screened many of them for
entomopathogenic activity. He reported that the
most effective was M. anisopliae. Screening of
over 100 isolates of this fungus has shown that
most isolates are pathogenic to termites and
that the most promising strains for biological
control were originally isolated from termites
or termite material (Milner et al., 1998b).
Recent field studies on Formosan termites have
identified promising strains of M. anisopliae
and Paecilomyces fumosoroseus which warrant
detailed field testing (Wright et al., 2005;
Meikle et al., 2005).
In Australia, a coelomic gregarine may be
clearly visible as cysts in the hemocoel of
workers of C. lacteus, but this protozoan has not
been shown to be pathogenic (Milner, unpub-
lished). Also, Gibbs et al. (1970) were able
to isolate virus-like particles from C. lacteus,
N. exitiosus and Porotermes adamsoni workers.
Again pathogenicity has not been demon-
strated. Nematodes such as Steinernema spp.
are not very effective pathogens in the
laboratory as the lethal concentration to kill
50% of worker termites is about 400 nematodes
(Wang et al., 2002; Mankowski et al., 2005).
In addition, they have not been reported
as natural pathogens. Ochiel (1995) isolated
the fungus Cordycepioideus bisporus (Pyreno-
mycetes: Clavicipitales) from alate adult
Macrotermes subhyalinus in Kenya and was able
to transmit the fungus to adults, but not to
workers, by exposing them to soil mixed with
in vitro produced ascospores and synnematal
material.
C Field testing of pathogens as microbial
insecticides against termites
There are very few reports of field-testing of
pathogens against termites. Ideally treatment
with a chemical or biological insecticide will
result in colony mortality. In practice, this
is difficult to achieve except where discrete
colonies are formed in mounds or trees. More
normally, treatment results in exclusion of
termites by physical barrier or repellency, or
treatment reduces the vigor of a colony for a
limited time necessitating repeat treatment.
Entomopathogenic nematodes such as
Steinernema spp. and Heterorhabditis spp. have
provided some limited control of termites under
field conditions, but have generally not proved
successful for long-term suppression (Maudlin
and Beal, 1989; Epsky and Capinera, 1988).
Hanel and Watson (1983) were able to introduce
M. anisopliae into nests of N. exitiosus by
applying conidia to feeding sites and while
some colonies were severely affected, others
showed little effect. They concluded that there
were unknown factors in the mound, which
inhibited conidial germination and so rendered
the pathogen dormant. The only successful direct
treatment of urban termites is that reported by
Milner et al. (1998b). The essential features of
this method for treating nests with conidia of
M. anisopliae are:
1. Locate the center of the nest by drilling into the
mound or suspect tree. Ideally a temperature probe
is used to ensure that the warmest part of the nest
(the nursery) is detected.
2. Apply the fungus in a dust preparation of pure dry
conidia, or conidia formulated in an inert carrier
such as talc, by blowing the material into the center
of the nest so as to ensure good distribution in
the nursery area. For a large nest, 5 g of pure
conidia has been found to be sufficient, assuming
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706 Milner and Pereira
a suitably virulent isolate of the fungus has been
selected. For nests in trees which do not have an
open system of galleries, it may be necessary to
apply down several holes to ensure good distri-
bution throughout the nursery area. A modified
double-action pump sold for inflating air-beds is
suitable for this type of application.
3. The effectiveness of the treatment can be evaluated
using a data logger to measure temperature
changes (Figure 2), or by sampling live termites
periodically and incubating them to assess level
of infection, or by destructive sampling of the
nest and checking for survival of the royal pair as
well as signs of cessation of reproduction. Infected
termites often move down from the center of the
colony and may be found, usually showing profuse
sporulation, at the base or even under the base of
the nest.
It is important to realize that live workers may
continue to be found in the nest and surrounding
feeding sites for many weeks after most of the
colony has been killed. However, depending
on the species of termites, nests can produce
neotenic queens (e.g., Coptotermes spp.) and
recover despite the loss of the royal pair.
Often the source nest for an infestation in
a building cannot be found or may be large
and diffuse as is the case with M. darwiniesis
and R. flavipes. In this case, the fungal dust
preparation can be applied in a similar manner
to that described for nests by blowing into
damaged timber such as skirting boards. Small
holes will need to be drilled at intervals of
about 1 m to ensure good distribution of the
conidia throughout the infested timber and the
conidial preparation blown in through these holes
using a plastic tube of similar diameter. It is
important that a large number of termites be
directly contacted by the conidia as it is likely
that after this initial kill, termites will be repelled
by the conidial deposit forcing them to feed
elsewhere. BioBlast
,aM. anisopliae product
that was sold by EcoScience in the USA for
termite control, was formulated as a wettable
powder. The product was mixed with water
and then pumped into feeding galleries where
termites were actively foraging. The aim of
treatment was claimed to be to transfer the
pathogen back to the nest; however, it may have
acted mainly as a repellent (Grace, 1997; Pearce,
1997). Wooden power poles, often used for trans-
mission of electricity in urban environments, may
be attacked by termites. A recent report on a
large field trial in New South Wales found that
blowing conidia into infested poles was effective
in controlling the termites. The fungus treatment
reduced the infestation rate by 70% after 6
months which was similar to that achieved by the
chemical insecticides, bifenthrin and triflumeron,
but it was less effective than dazomet which
was the most effective treatment giving 80%
control after 12 months. The life of the fungus
treatment was estimated as 22 months compared
with 37 months for dazomet (Martin Horwood,
pers comm., 2006). Metarhizium treatments may
also be effective in non-urban environments.
Figure 2. Temperature changes in a tree nest of Coptotermes acinaciformis following treatment with conidia of Metarhizium
anisopliae
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VII-20 Urban pests 707
For example Maniania et al. (2001) reported
that M. anisopliae applied to maize at planting
reduced termite damage and increased the yield
of grain. The effect was thought to be repellency
though some termite mortality may also have
occurred.
Methods for testing chemical insecticides as
soil treatments for termite controls are similar in
Australia and the USA and have been described
by Lenz et al. (1990) and Kard et al. (1989).
Similar methods were used by Milner et al.
(1993) to show that M. anisopliae conidia can
be applied to the soil surface, or mixed in with
soil, or painted as a suspension in Tween 80
onto timber surfaces, to protect a susceptible
piece of timber placed in the center. Soil surface
treatment with 8 ×10
7
conidia/cm
2
of isolate
FI-610 provided 100% protection for 24 weeks.
Depending on prevailing environmental condi-
tions this protection may last up to 3 years or
more (Milner and Staples, unpublished).
Pathogens have not been rigorously tested
for control of termites and so it is difficult to
be prescriptive. As slow acting toxicants, fungi
such as M. anisopliae are attractive for use in
baiting strategies to kill colonies. Consequently,
the methodology used by Su et al. (1996) to
evaluate baiting methods, could also be used with
pathogens. The essential features of this method
are the use of mark-recapture to assess population
density and to map the area of feeding by workers
from a single colony. A large number of bait
stations loaded with material such as susceptible
timber are then deployed in the ground over the
area. Once the termites start to feed at these
baits, they are replaced with similar baits laced
with a non-repellent formulation of a toxicant.
These toxic baits are replaced as needed and the
colony gradually declines. With hexaflumeron
(a benzophenylurea) it may take in excess of
9 months to reduce the colony to below the
detectable number of workers (Su et al., 1996).
Nile-blue is the most effective dye for labeling
termites (Evans, 1997) but some of the assump-
tions (e.g., random sampling) of standard mark-
recapture formulae are violated with termites
reducing the accuracy of this method for deter-
mining colony size (Forschler and Townsend,
1996; Thorne et al., 1996).
A major problem with using M. anisopliae
in baits containing conidia is the repellency
of the infectious conidia. Isolates vary in their
repellency and according to Begum and Jackson
(1994) the repellency is independent of virulence.
However, other workers have found that virulent
isolates can also be repellent (Delante et al.,
1995; Rath and Tidbury, 1997) though this
is dose-dependant (Milner and Staples, 1996).
Promising ways of overcoming this repellency
are to formulate the conidia in substrates such
as agar (Delante et al., 1995), organic amend-
ments (Milner, 2000), and cellulose (Wang and
Powell, 2004). These materials not only dilute the
conidia, but also may mask the factors inducing
repellency and are directly attractive for termites.
Ideally, candidate microbial products should
be tested on infested houses or structures.
However, as each example is different and it is
not possible to have controls, the results may
be impossible to interpret as the observed lack
of termite activity may be due simply to the
disturbance rather than the effect of the microbial
treatment. Krueger et al. (1995) reported that
of 101 structures treated with BioBlast™, 60%
were still free of further termite activity 6–15
months later. Similarly, Milner et al. (1993)
reported individual cases where M. anisopliae
had apparently given good control of infesta-
tions in houses for a period of time. While these
case histories and associated testimonials may
be useful for marketing a product, they cannot
constitute effective field testing, and therefore,
should be in support of scientific testing of the
types discussed in the preceding paragraphs.
D Conclusions
Urban termites present a unique challenge for
testing pathogens as they often do not have
a recognizable nest, have a complex behavior
linked with their social structure, and are difficult
to detect. Direct application to a nest will often
give complete control with a single application,
but this approach is not useful for termites such
as R. flavipes where the nest is diffuse. If the nest
is not affected, then the termites can continue
to reproduce at a high rate and replace the
individual termites killed by a localized appli-
cation of a pathogen. Consequently, damage may
continue or simply be transferred to another part
of the building. Future acceptable control by
pathogens may depend on either an integrated
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approach whereby the pathogen provides short-
term cessation of damage while changes are
made to the environment to make the building
less attractive to termites, alternatively pathogens
could be used in baits over a long period of time
to eliminate the colony. These approaches are the
subject of ongoing research.
5 References
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... Ant management with entomopathogens has mainly focused on their utilization as classical biological control agents to suppress invasive ant populations in landscapes surrounding buildings or in other outdoor habitats, which serve as reservoirs for invading ants ). While many pathogens and nematodes infecting ants have been reported, only a few have been the subject of extensive efforts to utilize them as MCAs (Evans, 1974;Oi and Pereira, 1993;Milner and Pereira, 2007;Evans et al., 2010Evans et al., , 2011. Nematodes and entomopathogens isolated from the red imported fire ant (S. invicta) have been the most extensively evaluated for their microbial control potential. ...
... An isolate (447) of B. bassiana was obtained from and shown to be virulent to red imported fire ants (Alves et al., 1988;Stimac et al., 1989;Stimac and Alves, 1994) before being directly applied to individual fire ant nests using different techniques and rates, but colony mortality was less than 50% (Oi et al., 1994). Bait formulations containing B. bassiana 447 or other isolates (Collins et al., 1999;Stimac and Pereira, 2001;Bextine and Thorvilson, 2002;Thorvilson et al., 2002;Milner and Pereira, 2007) have produced inconsistent results attributed to the limited intracolony transmission of fungus, microbial antagonism, and fungistatic action of fire ant venom (Storey et al., 1991;Pereira and Stimac, 1992;Oi and Pereira, 1993). Currently, B. bassiana is no longer being considered as an MCA for fire ants. ...
Chapter
Various reasons have prevented the microbial control of structural pests from reaching the commercial development and practical use achieved with agricultural pests. Microbial products have not been very successful on the market for the control of cockroaches throughout the world. The control of ants has focused on classical biological control agents used outdoors. Structural termite control has benefited little from work in agricultural systems, and doubts persist as to whether microbial control in structures can be successful. The recent resurgence of bed bugs has sparked an interest in the microbial control of these insects, but no commercial products are yet available. Without significant progress in the microbial control of the major structural pests, it is very unlikely that entomopathogens will become commercially available for the control of other structural pests. Due to the difficulties in fitting microbial products into structural pest management, monetary and time investments into the search and implementation of microbial control may lead to practical applications.
... There are few records of fungal infections that occur in wild cockroaches. The pathogenicity of fungal pathogens has not been deeply studied and is most of the time unknown (Milner and Pereira, 2007;Suiter, 1997). Montalva et al. (2016) studied the pathogenicity of M. blattodeae on the urban pest P. americana and found that this fungus caused 96 % mortality after 10 d. ...
Article
The aim of this study was to search for entomopathogenic fungi that infect wild cockroaches in forest ecosystems in two protected natural areas of Argentina. Two isolates of Metarhizium argentinense were obtained and identified from wild cockroaches (Blaberidae: Epilamprinae) through the use of morphological characteristics and molecular phylogenetic analyses. This novel species was found in Argentina and is a member of the Metarhizium flavoviride species complex. Phylogenetic analyses, based on sequence similarity analysis using internal transcribed spacer (ITS) and a set of four protein-coding marker sequences (EF1A, RPB1, RPB2 and BTUB), supported the status of this fungus as a new species. In addition, we tested the biological activity of the new species through assays against Blattella germanica nymphs and found that the two evaluated isolates were pathogenic. However, isolate CEP424 was more virulent and caused a confirmed mortality of 76 % with a median lethal time of 7.2 d. This study reports the southernmost worldwide location of a Metarhizium species that infects cockroaches and will help expand the knowledge of the biodiversity of pathogenic fungi of Argentine cockroaches.
... Metarhizium species are the leading bio-control agents well characterized regarding pathogenicity to agricultural [1,2], forest [3,4], public health [5], stored grains [6] and urban [7][8][9] insect pests. They infect the target host through the tight conidial adherence with the insect cuticle [10]. ...
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Metarhizium species are the leading bio-control agents well characterized regarding pathogenicity to agricultural, forest, public health, stored grains and urban insect pests. They infect the target host through the tight conidial adherence with the insect cuticle. Conidial binding to the insect cuticle drive the systematic integrated disease development events in target host to impart pathogenesis. However, there is growing evidence that virulence of the pathogen is directly related with proteolytic enzymes including metalloproteinases, chymotrypsin-like proteinases and subtilisin-like proteinases. Successful host pathogenesis is the selection of right set of virulence-related proteinases, which evolved as a result of host-pathogen coevolution.
... 4 The German cockroach, Blattella germanica (Dictyoptera: Blattellidae), is by far the most serious and predominant cosmopolitan pest in the world due to changes in human travel, commerce, and the urban environment. [5][6][7] Cockroaches have been detected around hospitals, sick rooms, areas of intensive care, surgical sections, etc. 8 Indeed, cockroaches are potential vectors of pathogenic organisms in the hospital environment. 9 In addition, numerous published papers recognized the association of cockroaches with several infectious diseases and the spread of drug-resistant microbes worldwide. ...
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Background: Cockroaches are among the most common pests in public dwellings and health facilities. Their presence can raise safety concerns, especially as they maybe carriers of pathogenic organisms. Methods: This study was carried out to isolate and identify the bacterial flora from German cockroaches (Blattella germanica). Cockroaches collected by hand catches from two public hospital environments in Tebessa city (northeast Algeria) were screened for microbial load from their external surfaces and alimentary tract using standard bacterial protocols. Results: A total of 174 bacterial isolates were isolated from 39 German cockroach specimens. The most common and abundant bacterial species belonged to the Pseudomonas group (23.5%) and Serratia (13.2%). Pathogens like Staphylococcus aureus were also isolated, as well as opportunistic pathogens like Klebsiella species and food spoilage bacteria such as Enterobacter and Citrobacter species were isolated from both external surface and digestive tract of the insect. Generalized linear models (GLM) were performed to analyze the variation of abundances and occurrences of bacterial isolates harboured by B. germanica. The GLMs revealed that the main factors affecting variation of bacterial diversity and abundance were sex and hospital (P < 0.001). Conclusion: The findings of this study suggest that German cockroach acts as reservoir and potential vector of some bacterial pathogens.
... Metarhizium is an insect-pathogenic fungus currently used as a biological control agent against various insect species (Lomer et al., 1997(Lomer et al., , 2001Milner & Pereira, 2000;Hunter et al., 2001;Maniania et al., 2003;Shah & Pell, 2003). Recent studies suggest that another ecological role of this fungus is as a plant rhizosphere associate. ...
Article
Here we tested the hypothesis that species of the soil-inhabiting insect-pathogenic fungus Metarhizium are not randomly distributed in soils but show plant-rhizosphere-specific associations. We isolated Metarhizium from plant roots at two sites in Ontario, Canada, sequenced the 5' EF-1α gene to discern Metarhizium species, and developed an RFLP test for rapid species identification. Results indicated a non-random association of three Metarhizium species (Metarhizium robertsii, Metarhizium brunneum and Metarhizium guizhouense) with the rhizosphere of certain types of plant species (identified to species and categorized as grasses, wildflowers, shrubs and trees). M. robertsii was the only species that was found associated with grass roots, suggesting a possible exclusion of M. brunneum and M. guizhouense. Supporting this, in vitro experiments showed that M. robertsii conidia germinated significantly better in Panicum virgatum (switchgrass) root exudate than did M. brunneum or M. guizhouense. M. guizhouense and M. brunneum only associated with wildflower rhizosphere when co-occurring with M. robertsii. With the exception of these co-occurrences, M. guizhouense was found to associate exclusively with the rhizosphere of tree species, predominantly Acer saccharum (sugar maple), while M. brunneum was found to associate exclusively with the rhizosphere of shrubs and trees. These associations demonstrate that different species of Metarhizium associate with specific plant types.
... It is possible that sporulation could be an important criterion for selection of fungal isolates for termite control. Termites are rarely infected naturally ( Milner, 1997;Milner and Pereira, 2000), mainly because they wall off or isolate the infected colony members ( Su et al., 1982;Zoberi, 1995). Quick sporulation after an infected termite dies may be advantageous in overcoming such defense behavior. ...
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Sporulation characteristics and virulence of Metarhizium anisopliae and Beauveria bassiana were examined in relation to laboratory transmission in Coptotermes formosanus. Fungal isolates significantly affected disease prevalence in termite populations. Sporulation of M. anisopliae played a more important role than virulence in producing epizootics within small groups of termites, but this was not the case for B. bassiana. Isolates characterized by quick sporulation (day 2 after death) did not exhibit better transmission in termites than those with high total sporulation (day 11 after death) in either fungal species. An isolate of M. anisopliae ranking highly in all three categories (virulence, quick sporulation, and total sporulation) produced better epizootics than an isolate that was inferior in all three characteristics. High temperatures (35 degrees C) significantly reduced fungal germination rates, leading to significant reduction of epizootics. M. anisopliae was better than B. bassiana in producing epizootics at 27 degrees C. Thus, fungal characteristics other than virulence should be considered for the seasonal colonization approach to termite microbial control.
... Sorokin var. acridum Milner & Driver, is being developed as a biopesticide for use against locusts and grasshoppers in Australia (Milner, 1997; Hunter et al., 1999; Milner and Pereire, 2000; Hunter et al., 2001) and Africa (Lomer et al., 2001). Development of similar biopesticides is being undertaken in many countries (Lomer et al., 2001) where locusts and grasshoppers are pests such as China (Lee et al., 2000), Brazil (Magalhaes et al., 2000), and Mexico (Hernández-velázquez and Gutierrez, 2000). ...
Article
The efficacy of a new virulent Metarhizium anisopliae variety (M. anisopliae var. dcjhyium, DQ288247) obtained from Odontotermes formosanus in China was evaluated against the subterranean termite, O. formosanus, in the laboratory. The new variety was compared with four other virulent M. anisopliae isolates and was found to be highly infectious and virulent against termites. M. anisopliae var. dcjhyium could cause approximately 100% mortality of termites 3 days post-inoculation in the concentration of 3x10(8) conidia/ml. There were also differences in relative hyhal growth and isoenzymes. M. anisopliae var. dcjhyium showed a different isoenzyme band pattern from the four isolates of M. anisopliae (AB027337, AB099510, AB099941 and AF280631). The phylogenetic tree of the 18S rDNA sequences revealed the taxonomic and evolutionary position of M. anisopliae var. dcjhyium. M. anisopliae var. dcjhyium and four isolates of M. anisopliae formed a monophyletic group, supported by a 99% bootstrap value. M. anisopliae var. dcjhyium formed a distinct variety, which had a special characterization of unique bands of isoenzyme, high virulence and low repellency against termites when compared with four other isolates of M. anisopliae.
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Phylogenetic analyses of insect-derived isolates of the Metarhizium anisopliae and M. flavoviride species complexes in Japan were conducted to reveal their species diversity. Fifty-seven isolates were identified as nine species, including one species first reported for Japan. Metarhizium pingshaense was the most frequently isolated species from this genus, and the 29 isolates of M. pingshaense came from six orders and 14 families of insects. New host–pathogen associations were found for two species with relatively narrow host ranges: Hymenoptera-M. pemphigi, Orthoptera- and Phasmatodea-M. majus.
Article
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Metarhizium anisopliae, the type species of the anamorph entomopathogenic genus Metarhizium, is currently composed of four varieties, including the type variety, and had been demonstrated to be closely related to M. taii, M. pingshaense and M. guizhouense. In this study we evaluate phylogenetic relationships within the M. anisopliae complex, identify monophyletic lineages and clarify the species taxonomy. To this end we have employed a multigene phylogenetic approach using near-complete sequences from nuclear encoded EF-1alpha, RPB1, RPB2 and beta-tubulin gene regions and evaluated the morphology of these taxa, including ex-type isolates whenever possible. The phylogenetic and in some cases morphological evidence supports the monophyly of nine terminal taxa in the M. anisopliae complex that we recognize as species. We propose to recognize at species rank M. anisopliae, M. guizhouense, M. pingshaense, M. acridum stat. nov., M. lepidiotae stat. nov. and M. majus stat. nov. In addition we describe the new species M. globosum and M. robertsii, resurrect the name M. brunneum and show that M. taii is a later synonym of M. guizhouense.
Article
Populations of 6 colonies of the Formosan subterranean termite, Coptotermesformosanus Shiraki, were significantly reduced but survived after 12-mo baiting using metabolic inhibitors such as A-9248 (diiodomethyl para-tolyl sulfone) or sulfluramid. These survived colonies recovered within several years and caused additional structural damage to the nearby buildings. Nine colonies (6 C. formosanus and 3 Reticulitermes flavipes (Kollar)) were eliminated after 2-9 mo baiting using the chitin synthesis inhibitor, hexaflumuron. Colony elimination generally created zones of termite-free soil that lasted for several years, except for one colony whose territory was invaded by a new C. formosanus colony 9 mo after the baiting. The presence of neighboring colony populations are evident in 3 sites but these neighboring colonies did not take over territories of eliminated colonies. Two additional colonies (one each of R. flavipes and C. formosanus) were intentionally left alive after partially suppression using hexaflumuron baits. One colony (R. flavipes) slowly declined and eventually collapsed 4 yrs after the baiting, while the other colony (C. formosanus) recovered. Results of this study demonstrated the advantages of colony elimination in providing long-term protection of structures from subterranean termites. Elimination of colony populations was achieved only when the chitin synthesis inhibitor, hexaflumuron, was used. Baits containing metabolic inhibitors such as A-9248 or sulfluramid only partially suppressed the colony populations even after the monthly placement of baits for 12-mo. Elimination of the vast populations of subterranean termite requires that the toxicant must be (1) slow-acting, (2) non-deterrent, (3) must not cause adverse effects when ingested at sublethal dose levels, and (4) its lethal time must be dose-independent.
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The National Home and Garden Pesticide Use Survey represents an attempt to compile data on the reasons why home pesticides are used, the extent of their use, and the methods used to apply, store, and dispose of them. The survey was conducted under contract to EPA by Research Triangle Institute. Data were collected by trained interviewers that visited each home personally. Data are available on specific pest problems, whether they are considered major or not, and whether the pests are managed in some way with pesticides. Other data are included on storage and disposal, use of child resistant packaging, and use of commercial pest control services.
Article
Metarhizium anisopliae DAT F-001 and ATCC 62176 were assayed against Coptotermes acinaciformis and Nasutitermes exitiosus. Spray application of 8.1 × 107 spores ml-1 of DAT F-001 and 1.2 × 108 spores ml-1 (ATCC 62176) for 3 seconds to the dorsal surface of the termites resulted in 100% mortality in 4 days. Unformulated spores were highly repellent to C. acinaciformis, but this repellency could be overcome by formulating the conidia in attapulgite clay (47%) and surfactant (3%). The spores could be made attractive to termites by the addition of further components to the formulation.