Pathogens of Insects

Pathogens are viruses or microorganisms that cause disease. Like all other organisms, insects are susceptible to a variety of diseases caused by pathogens. Many of these pathogens cause diseases that are acute and fatal and therefore are used as models to study processes of infection and pathogenesis as well as to control populations of insects that are pests or vectors of plant and animal diseases. Generally, insect pathogens have a relatively narrow host range and thus are considered to be more environmentally friendly than synthetic chemical insecticides. The pathogens that cause disease in insects fall into four main groups: viruses, bacteria, fungi, and protozoa. This article discusses the primary biological properties of each of these pathogen groups, with specific emphasis on how these pathogens have been used to benefit humans.

VIRUSES

Viruses are obligate intracellular parasites, meaning that they can reproduce only in living cells and are composed in the simplest form of a nucleic acid, either DNA or RNA, and a protein shell referred to as the capsid. More complex viruses also contain a lipoprotein envelope. Insect viruses can be cultured in living hosts (i n vivo) or in cultured insect cells (i n vitro). In general, insect viruses are divided into two broad nontaxonomic categories, the occluded viruses and the nonoc-cluded viruses. Occluded viruses are so named because after formation in infected cells, the mature virus particles (virions) are occluded within a protein matrix, forming paracrystalline bodies that are generically referred to as either inclusion or occlusion bodies. In the nonoccluded viruses, the virions occur freely or occasionally form paracrystalline arrays of virions that are also known as inclusion bodies. These, however, have no occlusion body protein interspersed among the virions.
The five most commonly encountered types of insect viruses are iridoviruses, cytoplasmic polyhedrosis viruses, entomopoxviruses, ascoviruses, and baculoviruses.


Iridoviruses

Nonoccluded viruses with a linear double-stranded DNA genome, the iridoviruses (family Iridoviridae) produce large, enveloped,icosahedral virions (125-200 nm) that replicate in the cytoplasm of a wide range of tissues in infected hosts. Virions form paracrystalline arrays in infected tissues, imparting an iridescent hue to infected hosts, from which the name of this virus group is derived. Over 30 types are known, and these have been most commonly reported from larval stages of Diptera larvae, such as mosquito larvae, as well as from larvae of Coleoptera and Lepidoptera. Generally, the irido-viruses occur very broadly, and they are known from other invertebrates, such as isopods, as well as from certain vertebrates including frogs and fish. Observations of natural occurrence in host field populations suggest that one host range of each type is quite narrow, although in the laboratory iridoviruses are readily transmitted from one insect species to another by inoculation. Prevalence and mortality rates in natural populations of host insects are typically less than 1%.

Cytoplasmic Polyhedrosis Viruses

The cytoplasmic polyhedrosis viruses (family Reoviridae) are occluded double-stranded RNA viruses with a genome divided into 9 or 10 segments of RNA. These viruses, commonly referred to as CPVs, cause a chronic disease and reproduce only in the stomach of insects, where typically they form large (ca. 0.5-2 |im) polyhedral to spherical occlusion bodies in the cytoplasm of midgut epithelial cells. Infection in early instars retards growth and development, extending the larval phase by weeks. The disease is often fatal. In advanced stages of disease, the infected midgut is white rather than translucent brown, because of large numbers of accumulated polyhedra. This virus type is relatively common among lepidopterous insects and among dipterous insects of the suborder Nematocera (e.g., mosquitoes, blackflies, midges). CPVs are typically easy to transmit by feeding to species that belong to the same family of the host from which they were isolated, and thus the host range of this virus type is quite broad.

Entomopoxviruses

The entomopoxviruses (family Poxviridae) are occluded double-stranded DNA viruses that produce large, enveloped virions (150 nm X 300 nm) that replicate in the cytoplasm of a wide range of tissues in most hosts, causing an acute, fatal disease. Occlusion bodies vary from being oval to spindle shaped and generally occlude 100 or more virions. These viruses have been most commonly reported from coleopterans, from which there are over 30 isolates, but they are also known from lepidopterous, dipterous (midges), and orthop-terous (grasshoppers) insects. This virus type is easily transmitted by feeding, although where the experimental host range of individual isolates has been tested, it has been found to be relatively narrow, generally being restricted to closely related species. Insect poxvi-ruses are related to vertebrate poxviruses, such as the variola virus, the etiological agent of smallpox, and they may be the evolutionary source of the vertebrate poxviruses.

Ascoviruses

The ascoviruses (Ascoviridae) are a new family of DNA viruses, at present known only from larvae of species in the lepidopteran family Noctuidae, where they have been reported from several common pest species such as the cabbage looper, cotton budworm, corn earworm, and fall armyworm. Ascoviruses cause a chronic, fatal disease of larvae. The virions of ascoviruses are large (130 nm X 400nm), enveloped, and reniform to bacilliform in shape; they exhibit complex symmetry and contain a circular, double-stranded DNA genome. During the course of ascovirus disease, large numbers of virion-containing vesicles accumulate in the blood of infected caterpillars, changing its color from translucent green to milky white. These virion-containing vesicles are formed by a unique developmental sequence resembling apoptosis (cell death) in which each infected host cell cleaves into a cluster of vesicles as virion assembly proceeds.
An interesting ascovirus feature is that transmission from host to host depends on vectoring by female endoparasitic wasps. Ascoviruses are very difficult to transmit by feeding, with typical infection rates averaging less than 15% even when larvae are fed thousands of vesicles in a single dose. In contrast, infection rates for caterpillars injected with as few as 10 virion-containing vesicles are typically greater than 90%, and experiments with parasitic wasps show that these insects can transmit ascoviruses.

Baculoviruses

Baculoviruses (family Baculoviridae) are large, enveloped, double-stranded, occluded DNA viruses. These viruses are divided into two main types, commonly known as the nuclear polyhedrosis viruses (NPVs) and the granulosis viruses (GVs). Both NPVs and GVs are highly infectious by feeding, and in some insect species periodically cause epizootics, that is, widespread outbreaks of disease, that result in significant (>90%) declines in caterpillar populations.

NUCLEAR POLYHEDROSIS VIRUSES

The NPVs (Fig. 1) are known from a wide range of insect orders but have been most commonly reported by far from lepidopterous insects, from which well over 500 isolates are known. Many of these are different viruses (i.e., viral species). NPVs replicate in the nuclei of cells, generally causing an acute fatal disease. The virions are large (80-200nm X 280nm) and consist of one or more rod-shaped nucleocapsids with a double-stranded circular DNA genome enclosed in an envelope. The occlusion bodies of NPVs are referred to commonly as polyhedra because typically their shape is polyhedral. Polyhedra are large (ca. 0.5-21 |im) and form in the nuclei, where each occludes as many as several hundred virions. The NPVs of lepidopterous insects infect a range of host tissues, but those of other orders are typically restricted to the midgut epithelium. Some NPVs have a very narrow host range and may replicate efficiently only in closely related species, whereas others, such as the AcMNPV (i.e., the NPV of the alfalfa looper, Autographa californica), have a relatively broad host range and are capable of infecting species in other genera.

GRANULOSIS VIRUSES

The GVs, of which over 100 isolates are known, are closely related to the NPVs but differ from the latter in several important respects. The virions of GVs are similar to those of NPVs but contain only one nucleocapsid per envelope. GVs are known only from lepidopterous insects. Like NPVs, they initially replicate in the cell nucleus, but pathogenesis involves early lysis of the nucleus (as virions begin to assemble), which in the NPVs occurs only after most polyhedra have formed. After the nucleus has lysed, GV replication continues throughout the cell, which now consists of a mixture of cytoplasm and nucleoplasm. When completely assembled, the virions are occluded individually in small (200 nm X 600 nm) occlusion bodies referred as granules. Many GVs primarily infect the fat body, whereas others have a broader tissue tropism and replicate throughout the epidermis, tracheal matrix, and fat body. One, the GV of the grapeleaf skeletonizer Harrisina bril-lians, is unusual in that it replicates only in the midgut epithelium.

Use of Viruses as Insect Control Agents

The best example of the use of a virus as an insect control agent is the use of the NPV of the European spruce sawfly, Gilpinia hercyniae,
Nuclear polyhedrosis virus polyhedra. (A) Wet mount preparation viewed with phase microscopy showing refractile poly-hedra in two infected nuclei. (B) Transmission electron micrograph through a single polyhedron showing the enveloped rod-shaped viri-ons, characteristic of NPVs, occluded within the polyhPathogens of Insectsedral matrix. Upon ingestion, this matrix dissolves in the insect midgut, and the virions invade the host through midgut microvilli.
FIGURE 1 Nuclear polyhedrosis virus polyhedra. (A) Wet mount preparation viewed with phase microscopy showing refractile poly-hedra in two infected nuclei. (B) Transmission electron micrograph through a single polyhedron showing the enveloped rod-shaped viri-ons, characteristic of NPVs, occluded within the polyhPathogens of Insectsedral matrix. Upon ingestion, this matrix dissolves in the insect midgut, and the virions invade the host through midgut microvilli.
as a classical biological control agent. The European spruce sawfly was introduced into eastern Canada from northern Europe around the turn of the last century and had become a severe forest pest by the 1930s. Hymenopteran parasitoids were introduced from Europe in the mid-1930s as part of a biological control effort, and inadvertently along with these came the NPV, which was first detected in 1936. Natural epizootics caused by the virus began in 1938, by which time the sawfly had spread over 31,000 km2. Most sawfly populations were reduced to below economic threshold levels by 1943 and remain under natural control today, the control being effected by a combination of the NPV, which accounts for more than 90% of the control, and the wasp parasitoids.
Although viruses, particularly NPVs, are frequently associated with rapid declines in the populations of important lepidopterous and hymenopterous (sawfly) pests, G. hercyniae NPV is the only example of a virus that has proven effective as a classical biological control agent. Another putative baculovirus, the “nonoccluded” bac-ulovirus of the palm rhinoceros beetle, Oryctes rhinoceros, has been a quasi-classical biological control success in that once introduced into populations in certain South Pacific islands, can yield control for several years, but ultimately it dissipates and must be reapplied. Moreover, augmentative seasonal introductions have been effective only rarely and are not well documented. Thus, the control potential of most viruses is best evaluated by assessing their utility as microbial insecticides. From this perspective, the iridoviruses are not effective because of their poor infectivity by feeding. Cytoplasmic polyhedro-sis viruses are not much better because, although highly infectious by feeding, the disease they cause is chronic. CPVs have, however, been useful in some situations, such as for suppression of the pine caterpillar, Dendrolimus spectabilis. in Japan. Ascoviruses and ento-mopoxviruses have not been developed as control agents for any insect owing to lack of efficacy.
For several reasons, the viruses most commonly used or considered as microbial insecticides in industrialized as well as less developed countries are the NPVs. First, NPVs are common in and easily isolated from pest populations. In addition, production in their hosts is cheap and easy, and the technology for formulation and application is simple and adaptable to standard pesticide application methods. Most NPVs, however, are narrow in their host range, infecting only a few closely related species. Furthermore, although several can be grown in vitro in small to moderate volumes (ca. 20- to 300 -l cell cultures), no fermentation technology currently exists for their mass production on a scale that would permit repeated applications to hundreds of thousands of acres, which is possible with Bacillus thur-ingiensis (Bt) chemical insecticides. These two key limitations have been major disincentives for the commercial development of NPVs, especially in industrialized countries.
Despite these drawbacks, several NPVs have been registered as microbial insecticides even though the market size for most is small. And registered or not, several are used in many less developed countries, particularly for control of lepidopteran pests of field and vegetable crops. Moreover, over the past decade there has been renewed interest in developing NPVs because recombinant DNA technology offers potential for improving the efficacy of these viruses.
In addition to the NPVs, a few GVs have also been successfully developed for pest control. These in the GVs of the codling moth and potato tuberworm moth, which are, respectively, serious pests of apples and potatoes in many regions of the world.

Other Uses

In addition to the use of NPVs in insect control, one baculovirus, the AcMNPV noted earlier, has been developed as an expression vector for producing a large number of foreign proteins in vitro. This expression system takes advantage of the strong polyhedrin promoter system, which in the wild-type viruses produces large amounts of the polyhedria used to occlude virions. By substituting foreign genes for the polyhedrin gene, it is possible to synthesize in insect cell cultures large quantities of foreign proteins, such as the capsid proteins of viruses that attack the vertebrates used for vaccine development and basic biomedical research.

BACTERIA

Bacteria are relatively simple unicellular microorganisms that lack internal organelles such as a nucleus and mitochondria, and reproduce by binary fission. With a few exceptions, most of those that cause disease in insects grow readily on a wide variety of inexpensive substrates, a characteristic that greatly facilitates their mass production. A variety of bacteria are capable of causing diseases in insects, but those that have received the most study are spore-forming bacilli (family Bacillaceae), especially B. thuringiensis. Many subspecies of Bt are used as bacterial insecticides and as a source of genes for insecticidal proteins added to make transgenic plants resistant to insect attack, especially attack by caterpillars and beetles. The other bacterial insect pathogens that have received various degrees of study are B. sphaericus, Paenibacillus popilliae, and P. larvae, the latter being the etiological agent of foulbrood, an important disease of honey bee larvae, Serratia entomophila and S. marcescens. Several of these, in order of importance, are discussed here to represent the diversity of bacteria that cause disease insects.

Bacillus thuringiensis

B. thuringiensis is a complex of bacterial subspecies that occur commonly in such habitats as soil, leaf litter, on the surfaces of leaves, in insect feces, and as a part of the flora in the midguts of many insect species. Bts are characterized by the production of a parasporal body during sporulation that contains one or more protein endotoxins in crystalline form (Fig. 2). Many of these are highly insecticidal to certain insect species. These endotoxins are actually protoxins activated by proteolytic cleavage in the insect midgut after ingestion. The activated toxins destroy midgut epithelial cells, killing sensitive insects within a day or two of ingestion. In insects species only moderately sensitive to the toxins, such as Spodoptera species (caterpillars commonly known as armyworms), the spore contributes to pathogenesis. Bt also produces other insecticidal compounds including (3-exotoxin, zwittermicin A, and vegetative insecticidal proteins (Vips).
The most widely used Bt is the HD1 isolate of B. thuringiensis subsp. kurstaki (Btk), an isolate that produces four major endo-toxin proteins packaged into the crystalline parasporal body (Fig. 2B). This isolate is the active ingredient in numerous commercially available bacterial insecticides used to control lepidopterous pests in field and vegetable crops, and in forests. Another successful Bt is the ONR60A isolate of B. thuringiensis subsp. israelensis (Bti), which is highly toxic to the larvae of many mosquito and blackfly species. This isolate also produces a parasporal body that contains four major endotoxins (Fig. 2B), but these are different from those that occur in Btk. Several commercial products based on Bti are available and are used to control both nuisance and vector mosquitoes and blackflies. Bti has proven to be an important environmentally compatible insecticide, and now has replaced the use of synthetic chemical insecticides for control of mosquito larvae in many countries around the world. A third isolate of Bt that has been developed commercially is the DSM2803 isolate of B. thuringiensis subsp. morrisoni (pathovar tenebrionis). This isolate produces a cuboidal parasporal body toxic to many coleopterous insects and is used commercially to control several beetle pests.
All the above-mentioned isolates are essentially used as bacterial insecticides, applied as needed. A variety of commercial formulations are available, including emulsifiable concentrates, wettable powders, and granules, for use against different pests in a variety of habitats. On a worldwide basis, millions of hectares are treated annually with products based on Bt. Estimates indicate the worldwide market is about $80-100 million. Although used as a bacterial insecticide, plants have been engineered to produce Bt Cry (crystal) proteins for resistance to insects, and this use has now far surpassed the use of Bt insecticides worldwide. Crops that produce Bt Cry proteins against lepidopterous and coleopterous pests, such as Bt cotton and Bt maize are now used in many countries including the United States, China, India, and Argentina, where the total market value now is several billions of dollars per year. This technological development has proven of great environmental benefit in that it has reduced the use of synthetic chemical insecticides by millions of pounds per year. As the technology of Bt crops is improved and public acceptance expands, Bt crops will become commonly used in many other countries during this century.

Bacillus sphaericus

Since the mid-1960s it has been known that many isolates of B. sphaericus (Bs) are toxic to certain mosquito species. Over the past three decades, three isolates have been evaluated for their mosquito
 Sporulating cell of Bacillus thuringiensis and insec-ticidal parasporal bodies. (A) Transmission electron micrograph through a cell of B. thuringiensis subsp. israelensis illustrating a developing spore (Sp) and endotoxin-containing parasporal body (PB) outside the exoporium membrane, E. Bar, 250 nm. (B) Scanning electron micrograph of parasporal bodies (crystals) of B. thuringiensis subsp. kurstaki, a subspecies used widely to control caterpillar pests. The bipyramidal crystals contain three endotoxins (Cry1Aa, Cry1Ab, and Cry1Ac), whereas the smaller cuboidal crystal contains a single endotoxin (Cry2A). The bipyramidal crystals contain three endotoxin proteins (Cry1Aa, Cry1Ab, and Cry1Ac), and the cuboidal crystal has an additional toxin (Cry2A). This toxin complexity accounts for the broad spectrum of activity of many isolates of B. thuringiensis subsp. kurstaki. (C) Transmission electron micrograph of a parasporal body of B. thuringiensis subsp. israelensis used widely to control the larvae of mosquitoes and blackflies. This parasporal body is also composed of four major endotoxins, a large semispherical inclusion containing Cyt1Aa, a dense spherical body that apparently contains the Cry4Aa and Cry4Ba proteins, and a bar-shaped body that contains Cry11Aa. The endotoxin inclusions of this subspecies are held together by an envelope of unknown composition. This parasporal body has the highest specific toxicity of known Bt species, and this is due to synergistic interactions between the Cyt1Aa and Cry proteins as well as synergistic interactions among the Cry proteins. Bt endotoxins act by destroying the insect midgut epithelium (stomach).
FIGURE 2 Sporulating cell of Bacillus thuringiensis and insec-ticidal parasporal bodies. (A) Transmission electron micrograph through a cell of B. thuringiensis subsp. israelensis illustrating a developing spore (Sp) and endotoxin-containing parasporal body (PB) outside the exoporium membrane, E. Bar, 250 nm. (B) Scanning electron micrograph of parasporal bodies (crystals) of B. thuringiensis subsp. kurstaki, a subspecies used widely to control caterpillar pests. The bipyramidal crystals contain three endotoxins (Cry1Aa, Cry1Ab, and Cry1Ac), whereas the smaller cuboidal crystal contains a single endotoxin (Cry2A). The bipyramidal crystals contain three endotoxin proteins (Cry1Aa, Cry1Ab, and Cry1Ac), and the cuboidal crystal has an additional toxin (Cry2A). This toxin complexity accounts for the broad spectrum of activity of many isolates of B. thuringiensis subsp. kurstaki. (C) Transmission electron micrograph of a parasporal body of B. thuringiensis subsp. israelensis used widely to control the larvae of mosquitoes and blackflies. This parasporal body is also composed of four major endotoxins, a large semispherical inclusion containing Cyt1Aa, a dense spherical body that apparently contains the Cry4Aa and Cry4Ba proteins, and a bar-shaped body that contains Cry11Aa. The endotoxin inclusions of this subspecies are held together by an envelope of unknown composition. This parasporal body has the highest specific toxicity of known Bt species, and this is due to synergistic interactions between the Cyt1Aa and Cry proteins as well as synergistic interactions among the Cry proteins. Bt endotoxins act by destroying the insect midgut epithelium (stomach).
control potential, 1593 from Indonesia, 2297 from Sri Lanka, and 2362 from Nigeria. The 1593 and 2297 isolates were obtained form soil and water samples at mosquito breeding sites, whereas 1593 was isolated from a dead adult blackfly.
Like Bt, Bs acquires its toxicity as the result of protein endotox-ins that are produced during sporulation and assembled into par-asporal bodies. Bs is unusual in that the main toxin is a binary toxin (i.e., composed of two protein subunits). These are proteolytically activated in the mosquito midgut to release peptides having molecular masses of, respectively, 43 and 39 kDa, that associate to form the binary toxin, with the former protein constituting the binding domain, and the latter the toxin domain. The toxins bind to micro-villi of the midgut epithelium, causing hypertrophy and lysis of cells, destroying the midgut and killing the mosquito larva.

Paenibacillus popilliae

P. popilliae is a highly fastidious bacterium that is the primary eti-ological agent of the so-called milky diseases of scarab larvae. These insects are the immature stages of beetles, such as the Japanese beetle, Popillia japonica, that are important grass and plant pests belonging to the coleopteran family Scarabaeidae. The term “milky disease” is derived from the opaque white color that characterizes diseased larvae and results from the accumulation of sporulating bacteria in larval hemolymph (blood). The disease is initiated when grubs feeding on the roots of grasses or other plants ingest the bacterial spores. The spores germinate in the midgut and vegetative cells invade the midgut epithelium, where they grow and reproduce, changing in form as they progress toward invasion of the hemocoel (body cavity). After passing through the basement membrane of the midgut, the bacteria colonize the blood over a period of several weeks and sporulate, reaching populations of 100,000,000 cells ml-1. For larvae that ingest a sufficient number of spores early in development, the disease is fatal. Dead larvae in essence become foci of spores that serve as a source of infection for up to 30 years.
Despite decades of research, suitable media for the growth and mass production of P. popilliae in vitro have not been developed. Thus, the technical material (i.e., spores) used in commercial formulations is produced in living, field-collected scarab larvae. Nevertheless, a small but steady market remains for P. popilliae in the United States because of serious problems due to scarab larvae, such as damage to turf grass by larvae of the Japanese beetle.

Serratia entomophila

A novel bacterium named. S. entomophila causes amber disease in the grass grub, Costelystra zealandica. an important pest of pastures in New Zealand, and has been developed as a biological control agent for this pest. This bacterium adheres to the chitinous intima of the foregut, where it grows extensively, eventually causing the larvae to develop an amber color; the result of infection is death. The bacterium is easily grown and mass-produced i n vitro and can now be grown to densities as high as 4 X 1010cellsml_1. Successful mass production of S. entomophila led to its rapid commercialization. It is now used to treat infested pastures in New Zealand at a rate of one liter of product per hectare. Liquid formulations of this living, nonspore-forming bacterium are applied with subsurface application equipment. The rapid development and commercialization of the bacterium, even though the use is rather restricted, shows how microbials can be successful in niche markets, where there are few alternatives, and mass production methods, the most critical factor, are available.

FUNGI

The fungi constitute a large and diverse group of eukaryotic organisms distinguished from others by the presence of a cell wall, as in plants, but lacking chloroplasts and thus the ability to carry out photosynthesis. Fungi live either as saprophytes or as parasites of plants and animals, and require organic food for growth, obtained by absorption from the substrates on which they live. The vegetative phase, known as a thallus, can be either unicellular, as in yeasts, or multicellular and filamentous, forming a mycelium, the latter being characteristic for most of the fungi that attack insects. During vegetative growth, the mycelium consists primarily of hyphae, which may be septate or nonseptate, and these grow throughout the substrate to acquire nutrients. Reproduction can be sexual or asexual, and during this phase the mycelium produces specialized structures such as motile spores, sporangia, and conidia, typically the agents by which fungi infect insects. Fungi usually grow best under wet or moist conditions, and those that are saprophytic as well as many of the parasitic species are easily cultured on artificial media.
The fungi are divided into five major subdivisions, and these reflect the evolution of the biology of fungi from aquatic to terrestrial habitats. For example, species of the genera Coelomomyces and Lagenidium (subdivision Mastigomycotina) are aquatic and produce motile zoospores during reproduction, whereas members of the genera Metarhizium and Beauveria (subdivision Deuteromycotina) are terrestrial and reproduce and disseminate via nonmotile conidia.
Unlike most other pathogens, fungi usually infect insects by active penetration through the cuticle. The typical life cycle begins when a spore, either a motile spore or a conidium, lands on the cuticle of an insect. Soon after, under suitable conditions, the spore germinates, producing a germ tube that grows and penetrates down through the cuticle into the hemocoel. Once in the hemolymph, the fungus colonizes the insect. Hyphal bodies bud off from the penetrant hyphae and either continue to grow and divide in a yeastlike manner or elongate, forming hyphae that grow throughout the insect body. Complete colonization of the body typically requires 7-10 days, after which the insect dies. Some fungi produce peptide toxins during vegetative growth, and in these strains death can occur within 48 h. Subsequently, if conditions are favorable, which generally means an ambient relative humidity of greater than 90% in the immediate vicinity of the dead insect, the mycelium will form reproductive structures and spores, thereby completing the life cycle. Depending on the type of fungus and species, these will be produced either internally or externally as motile spores, resistant spores, sporangia, or conidia.
Fungi are one of the most common types of pathogens observed to cause disease in insects in the field. Moreover, outbreaks of fungal diseases under favorable conditions often lead to spectacular epizootics that decimate populations of specific insects over areas as large as several hundred square kilometers. As a result, there has been interest in using fungi to control insects for well over a century; the first efforts, in Russia in the late 1880s, used Metarhizium anisopliae to control the wheat cockchafer Anisoplia austriaca. Although there have been numerous attempts since then to develop fungi as commercial microbial insecticides, very few of these efforts have met with success. Thus, at present barely a handful of commercially available fungal insecticides are available for use in industrialized countries, and true commercial success has remained elusive. On the other hand, in developing countries (e.g., Brazil and China), “cottage industry” technology like that used to produce viruses has been turned to the production fungi such as M. anisopliae and Beauveria bassiana. A quasi-commerical product Boverin, developed and used in Russia
for control of the Colorado potato beetle, proved ineffective in the United States. Current efforts to find alternatives to chemical insecticides have intensified research on fungi, with the aim of identifying new isolates or improving existing strains through molecular genetic manipulation. Over the past decade, several isolates of M. anisopliae, for example, have been used to control orthopteran (locust) pests in Africa and the Middle East, and the use of strains of this species to control the mosquito vectors of malaria are underway in Africa. Researchers hope to obtain products that will prove more successful as either classical biological control agents or mycoinsecticides. The subsections that follow summarize briefly the critical biological features of selected fungi to illustrate the advantages and disadvantages of these as control agents.

Aquatic Fungi

Aquatic fungi of two types that attack mosquito larvae have received considerable study: species of Coelomomyces (class Chytridiomycetes: order Blastocladiales) and Lagenidium giganteum (class Oomycetes: order Lagenidiales).
The genus Coelomomyces comprises over 80 species of obligately parasitic fungi that have a complex life cycle involving an alternation of sexual (gametophytic) and asexual (sporophytic) generations. The sexual phase parasitizes a microcrustacean host, typically a cope-pod, whereas the asexual generation develops, with rare exception, in mosquito larvae. In the life cycle, a biflagellate zygospore invades the hemocoel of a mosquito larva, where it produces a sporophyte that colonizes the body and forms resistant sporangia. The larva dies and subsequently the sporangia undergo meiosis, producing uni-flagellate meiospores that invade the hemocoel of a copepod host, where a gametophyte develops. At maturation, the gametophyte cleaves, forming thousands of uniflagellate gametes. Cleavage results in death of the copepod and escape of the gametes, which complete the life cycle by fusing to biflagellate zygospores, which then seek out another mosquito host. The life cycles of these fungi are highly adapted to those of their hosts. Moreover, as obligate parasites these fungi are very fastidious in their nutritional requirements, and as a result no species of Coelomomyces has been cultured in vitro.
Coelomomyces, the largest genus of insect-parasitic fungi, has been reported worldwide from numerous mosquito species, many of which are vectors of important diseases such as malaria and filiaria-sis. In some of these species, Anopheles gambiae in Africa, for example, epizootics caused in some areas by Coelomomyces kill greater than 95% of the larval populations. Such epizootics led to efforts to develop several species as biological control agents. For several reasons, however, these efforts were discontinued. One important factor was the discovery that the life cycle requires a second host for completion. Also contributing were the inability to culture these fungi in vitro and the development of Bti as a bacterial larvicide for mosquitoes.
Although it is unlikely that Coelomomyces fungi will be developed as biological control agents, interest remains in developing L. giganteum. This oomycete fungus is easily cultured on artificial media and does not require an alternate host. In the life cycle, a motile zoospore invades a mosquito larva through the cuticle. Once within the hemocoel, the fungus colonizes the body over a period of 2-3 days, producing an extensive mycelium consisting largely of non-septate hyphae. Toward the end of growth, the hyphae become septate, and out of each segment an exit tube forms which grows back out through the cuticle and forms zoosporangia at the tip. Zoospores quickly differentiate in these, exiting through an apical pore to seek out a new substrate. In addition to this asexual cycle, thick-walled resistant sexual oospores can be formed in the mosquito cadaver.

Terrestrial Fungi

The fungi that have received the most attention for use in biological control are terrestrial fungi, with most emphasis placed on the development of selected species of hyphomycetes such as M. anisopliae and B. bassiana for use as microbial insecticides. In addition, the more specific and nutritionally fastidious entomophthoraceous fungi continue to receive attention, but for their potential use as classical biological control agents rather than as microbial insecticides. Representative examples of these terrestrial fungi are discussed in the subsections that follow.

ENTOMOPHTHORALES

These fungi comprise a large order of the class Zygomycetes that contains numerous genera, many species of which are commonly found parasitizing insects and other arthropods. The fungi routinely cause localized and sometimes widespread epizootics in populations of hemipterous and homopterous insects, particularly aphids and leafhoppers, but also in insects of other types such as grasshoppers, flies, beetle larvae, and caterpillars. In addition, a few species of the genus Conidiobolus are able to cause mycoses in some mammals, including humans. Apart from these few species, most of the entomophthoraceous fungi are highly specific, obligate parasites of insects and therefore their use for biological control poses no threat to nontarget organisms. As with Coelomomyces, however, the complex nutritional requirements, which have thus far prevented mass production in vitro, and high degree of host specificity, make these fungi poor candidates for development as microbial insecticides. Moreover, the conidia are very fragile, providing a challenge to formulation, and the resistant spores, like the oospores of L. giganteum, are difficult to germinate in a predictable manner. Nevertheless, there is evidence that if cultural practices in crop production are modified, these fungi can provide effective insect control where they occur naturally, and through the introduction of foreign strains and species (i.e., a classical biological control approach).
The most important genera found attacking insects in the field are Conidiobolus (aphids), Erynia (aphids), Entomophthora (aphids), Zoophthora (aphids, caterpillars, beetles), and Entomophaga (grasshoppers, caterpillars). Although many species of these genera cause epizootics and have received considerable study, none really seems to have much potential for development as a commercial microbial insecticide. On the other hand, cultural control, classical biological control, and environmental monitoring methods continue to show promise for using entomophthoraceous fungi for insect control. For example, the introduction of Erynia radicans from Israel into Australia to control the spotted alfalfa aphid, Therioaphis maculata, has proven a classical biological control success.
A relatively recent example of apparent classical biological control can he found in the natural outbreaks of Entomophaga maimaiga in larval populations of the gypsy moth, Lymantria dispar, an important pest of deciduous forests throughout several states comprising the middle Atlantic and New England regions of the United States. Outbreaks of E. maimaiga have reduced larval populations to below economic thresholds, and the fungus is spreading westward naturally, and with human assistance, to gypsy moth populations established in other states. The source of this fungus is Japan, although it is not clear when the fungus causing present outbreaks of disease first appeared in the United States. The fungus was purposely introduced into the United States around the turn of the century, but seems not to have become established at that time. Then in the late 1980s, outbreaks of E. maimaiga began to occur in Connecticut and New York, and later in Virginia. In areas where it has established, given sufficient rainfall, the fungus seems to be capable of keeping the gypsy moth population below defoliation levels. It will require another 10 years of evaluation to determine whether this is a valid instance of classical biological control by a fungus.

CLASS HYPHOMYCETES

The hyphomycete fungi belong to the fungal subdivision Deuteromycotina (imperfect fungi), a grouping erected to accommodate fungi for which the sexual phase (perfect state) has been lost or remains unknown. This group contains the fungal species that most workers consider to have the best potential for development as microbial insecticides, B. bassiana and M. anisopliae, the agents of, respectively, the white and green mus-cardine diseases of insects. Unlike the fungi already discussed, these two species have very broad host ranges and probably are capable of infecting insects of most orders.
With respect to the general life cycle of these fungi, the process of invasion, colonization of the insect body, and formation of conidi-ophores and conidia is similar to that described for the other fungi. During invasion and colonization, some fungal species produce peptide toxins that quicken host death. The infectious agent is the conidium (Fig. 3), and the taxonomy for the hyphomycetes is based primarily on the morphology of the reproductive structures, particularly the conidiophores and the conidia. Most of the hyphomycete fungi used or under development grow well on a variety of artificial media, and this attribute, along with their ability to infect insects via the cuticle, favors commercial development. In the “cottage industry” commercial operations in Brazil, China, and the former Soviet Union, solid or semisolid substrates are used for production, and the primary ingredients are grain or grain hulls.
In general, the development of B. bassiana and M. anisopliae is being targeted for control of insects that live in cooler and moist environments, such as beetle larvae in soil and planthoppers on rice, though the former species is also being evaluated against whiteflies in glasshouses, as well as grasshoppers, especially locusts, in field crops. In addition to these two species, several species with much narrower host ranges are considered to have potential for development, including Paecilomyces fumoso-rosea (for whiteflies), Verticillium lecanii (for aphids and whiteflies in glasshouses), Hirutella thompso-nii (for mites), and Nomurea rileyi (for noctuid caterpillars).
With these apparent advantages, it is natural to ask why none of the hyphomycete fungi have been commercially successful as micro-bial insecticides in developed countries. There are several reasons related to their biological properties. First and foremost is that the production of conidia or mycelial fragments that are used as the active ingredient of formulations is not cost-effective because too much material is required to allow the achievement of an acceptable level of control. In addition to the problem of inefficient yields, the formulations are bulky, and preservation of fungal viability beyond a few months is low because the conidia are fragile. In mosquito and blackfly control, similar constraints apply. In addition, the discovery of cost-effective strains of B. thuringiensis and B. sphaericus has generally eliminated imperfect fungi, as well as many other microorganisms, for consideration as biological control agents for these important nuisance and vector insects.
In developing countries, B. bassiana and M. anisopliae were used in the past in some crops with considerable success. For example, in China, B. bassiana was used to control the European corn borer and Ostrinia nubilalis in maize. The fungus was produced in large covered pits on maize stalks. In Brazil, a preparation of M. anisopliae known
Typical reproductive structures of deuteromycete (imperfect) fungi. (A-D) Wet mount preparation of conidia-generating cells and conidia of Verticillium lecanii, which commonly attacks aphids and whiteflies. The conidia visible as free conidia and conidial clusters in (B) and (D) are the principal infective units. When these come in contact with an insect host, they germinate and penetrate the body, forming a mycelium that colonizes the insect over a period of several days. When conditions are appropriate, typically meaning high relative humidity, hyphae penetrate back out through the cuticle, producing conidiophores, the visible branched structures in these panels (A-C), which form reproductive conidia at their tips.
FIGURE 3 Typical reproductive structures of deuteromycete (imperfect) fungi. (A-D) Wet mount preparation of conidia-generating cells and conidia of Verticillium lecanii, which commonly attacks aphids and whiteflies. The conidia visible as free conidia and conidial clusters in (B) and (D) are the principal infective units. When these come in contact with an insect host, they germinate and penetrate the body, forming a mycelium that colonizes the insect over a period of several days. When conditions are appropriate, typically meaning high relative humidity, hyphae penetrate back out through the cuticle, producing conidiophores, the visible branched structures in these panels (A-C), which form reproductive conidia at their tips.
as Metaquino has been used for many years on sugarcane plantations and in pastures to control the spittlebug, Mahanarva posticata. Fungal conidia are produced in sealed plastic bags on rice. Figures indicate that as many as 50,000 ha are treated annually, and reductions in spittlebug populations are sufficient to keep populations below damaging levels. In the South Pacific, M. anisopliae has also been used to assist control of the rhinoceros beetle, Oryctes rhinoceros, a serious pest of coconut palms. Application of conidia at a rate of 50 gm~2 of soil yielded 80% larval mortality and improved coconut yields by 25%. Although these are examples of local successes, their applicability to agricultural production in developed countries is questionable. Moreover, the use of Bt crops such as Bt maize, noted above, has proven more effective than using fungi for control of important lepidopterous and coleopterous pests. Thus, in the future, genetic engineering techniques are likely to dominate crop development where insecticidal proteins are available to control the most important pests. In other situations, however, fungi such as M. anisopliae and B. bassiana may prove particularly useful. Examples include the control of sucking insect pests like aphids, white flies, and sharpshooters, or insects such as locusts and adult mosquitoes, where other effective pathogens are not available. As with bacterial insecticides, these fungi are amenable to genetic engineering techniques. It has already been shown recently in the case of M. anisopliae that its insecticidal efficacy can be improved by adding insecticidal proteins and enzymes to the battery of virulence agents that it produces during the process of infecting insects.


PROTOZOA

Protozoa is a general term applied to a large and diverse group of eukaryotic unicellular motile microorganisms that belong to what is now known as the kingdom Protista. Members of this kingdom can be free-living and saprophytic, commensal, symbiotic, or parasitic. The cell contains a variety of organelles, but no cell wall, and cells vary greatly in size and shape among different species. Feeding is by ingestion or more typically by adsorption, and vegetative reproduction is by binary or multiple fission. Sexual reproduction, often useful for taxonomy, can be very complex, but asexual reproduction occurs as well. Many protozoa produce a resistant spore stage that is also used in taxonomy. Divided into a series of phyla based primarily on mode of locomotion and structure of locomotory organelles, the kingdom includes the Sarcomastigophora (flagellates and amoebae), Apicomplexa (sporozoa), Microspora (microsporidia), Acetospora (haplosporidia, now thought to be a type of parasitic alga), and Ciliophora (ciliates). Protozoa of some types, such as the free-living amoebae and ciliates, are easily cultured in vitro, whereas many of the obligate intracellular parasites have not yet been grown outside cells.
As might be expected from such a large and diverse group of organisms, many species of protozoans are associated with insects, and the biology of these associations covers the gamut from being symbiotic to parasitic. Those that are parasitic have the general feature of causing diseases that are chronic. Many of the parasitic types, especially the microsporidia, build up slowly in insect populations, eventually causing epizootics that lead to rapid declines in populations of specific species. These epizootics attracted interest in the possibility of using protozoa to control pest insects, and over the past several decades numerous studies have been aimed at evaluating this potential. In general, these studies have shown that protozoa hold little potential for use as fast-acting microbial insecticides because of the chronic nature of the diseases they cause and because commercially suitable methods for mass production are lacking. However, as with the entomophthoraceous fungi, the possibility exists that protozoans, particularly microsporidia, may be useful as classical biological control agents. Clear examples of the effectiveness of such strategies remain to be demonstrated.
The life cycles and biologies that occur among the various protozoa that attack insects are too diverse in relation to their pest control potential for even a few to be covered here. Instead, the group with the most potential—the microsporidia—is described in terms of general biology and possible use in insect control.

General Biology of Microsporidia

The microsporidia (phylum Microspora) are the most common and best studied of the protozoans that cause important diseases of insects. Although still referred to as protozoa, recent molecular phylogen-tic studies have shown that these pathogens evolved from fungi. Well over a 1000 species are known, and most of these have been described from insects . Microsporidia have been most commonly described from insects of the orders Coleoptera, Lepidoptera, Diptera, and Orthoptera, but they are also known from other orders and probably occur in all. The epizootics in insect populations caused by protozoa are usually due to microsporidia. All microsporidia are obligate intracellular parasites and are unusual in that they lack mitochondria. In addition, they produce spores that are distinguished from the spores of organisms of all other known types by the presence of a polar filament (Fig. 4), a long coiled tube inside the spore used to infect hosts with the sporoplasm.
The typical microsporidian life cycle begins with the ingestion of the spore by a susceptible insect. Once inside the midgut, the polar filament everts, rapidly injecting the sporoplasm into host tissue.
Representative microsporidian spore: transmission electron micrograph through a uninucleate spore of Amblyospora abserrati from a larva of the mosquito Ochlerotatus abserratus. The circular structures on each side of the spore are cross sections through the polar filament that is used to inject the contents of the spore into the mosquito body after ingestion and activation of the spore.
FIGURE 4 Representative microsporidian spore: transmission electron micrograph through a uninucleate spore of Amblyospora abserrati from a larva of the mosquito Ochlerotatus abserratus. The circular structures on each side of the spore are cross sections through the polar filament that is used to inject the contents of the spore into the mosquito body after ingestion and activation of the spore.
The sporoplasm is unicellular but may be uni- or binucleate. Upon entry into the cytoplasm of a host cell (e.g., the fat body in many species of insects), the sporoplasm forms a plasmodium (meront), which undergoes numerous cycles of vegetative growth (merogony). During these cycles, the cells multiply extensively, dividing by binary or multiple fission and spreading to other cells, and, in many species, to other tissues of the host. After several merogonic cycles, the microsporidian undergoes sporulation. This consists of two major phases, sporogony—a terminal reproductive division committed to sporulation—and spore morphogenesis. In the sexual phase of reproduction, meiosis occurs early during sporogony. The spores, which in general measure several micrometers in diameter and length, have a thick wall and are highly, refractile when viewed by phase microscopy. The disease often lasts for several weeks during which billions of spores may accumulate in the tissues of a single infected host.
Microsporidian systematics is based on the size and structure of the spores, life cycles, and host associations. In addition to transmission by ingestion, many microsporidia are transmitted vertically from adult females to larvae via the egg (transovarially). With respect to host range, some species are species specific, whereas others occur in many species of the same family or order, and some can be transmitted to insects of different orders.

Microsporidia as Biological Control Agents

Naturally occurring epizootics caused by microsporidia are periodically very effective in significantly reducing insect pest populations. The problem is that these epizootics cannot be predicted with any degree of accuracy, nor can they be relied upon for adequate control, even though many of the conditions that facilitate their occurrence are known. The epizootics caused by Nosema pyrausta in populations of the European corn borer are often a classic example of this unreliability. These epizootics are useful when they occur, but because this often happens too late to prevent economic damage; reliance on N. pyrausta alone is insufficient. Thus, efforts have been directed toward developing methods for amplifying spore loads in the field through inundative releases, in essence using microsporidia as microbial insecticides.
Because they are obligate intracellular parasites that lack mitochondria, microsporidia cannot be grown on artificial media. Several species have been grown, however, in established insect cell lines, although this is not practical for field use. For field application, whether for microbial insecticide trials or for introductions into populations, spores are grown in living hosts. With such methods the yield can be quite high (109-1010 spores per host). These yields in terms of the number of larvae that must be grown to treat a hectare and infect most of the target population are comparable to the requirements for nuclear polyhedrosis viruses. Thus, if the microsporidia could cause acute diseases, they would be on an equal footing with many of the NPVs. However, the diseases are chronic, and even if a high percentage of the target pest population is infected, there all too often is little, if any, crop protection. In fact, if advanced instars such as thirds and fourths are treated, the larvae may live longer and cause greater crop damage than if the fields were left untreated. Thus, microsporidia are not useful as microbial insecticides.
There is now a general realization that microsporidia and other protozoans have virtually no potential for use as microbial insecticides. They may, however, be useful as population management tools.

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