Bacteria as insect pathogens.

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BACTERIA AS INSECT

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Annu. Rev. Microbiol. 1975.29:163-190. Downloaded from www.annualreviews.org by University of Minnesota - Twin Cities on 08/05/13. For personal use only.

PATHOGENS Lee A. Bulla Jr. u.s. Grain Marketing Research Center. ARS. USDA. Manhattan. Kansas 66502

Robert A. Rhodes and Grant St. Julian Northern Regional Research �enter. ARS. USDA. Peoria. Illinois 61604

CONTENTS INTRODUCTION

163

COMPARISON OF BACTERIA TO OTHER MICROBIAL PATHOGENS ..............

164

SYSTEMATICS AND TAXONOMy ...............................................

167

Classification and Identification ...........................................

167

. . ...... . ... .

170

Growth Characteristics ... ....... . ... . ... . ... . ............................

172

Host-Pathogen Relationships and Specificities .. PHYSIOLOGY Nutritional ReqUirements

171

. . . . .

. . . .. . . . .

. .

..

. .

.. ...

. .

.. . . . . . .... . .. . . .. . . .

Unique Metabolic Properties ..............................................

Secondary Metabolism and End-Producr Synthesis ................ ............

173 176 178

PATHOLOGy ................................................................... Basis of Pathogenicity invasion Routes ..... . ..................................................

Host-Pathogen Interactions

. . .

. . .. .

. .

.. . .. . .... . . . ...... . .... . .. . ........ ,'

182 183

USE OF BACTERIA AS INSECTICIDES ........................................ ,.. Basic Requirements .....................................................

184

Experimental and Field Applications .......................................

185

Safety and Environmental Implications . . ....... , .. . .......... . .............

186

CONCLUSIONS

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181

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INTRODUCTION Maladies of insects have been observed since the third century. Ar istotle (6) de scribed diseases of insects in his writings. He specifically denoted a sickness of the honey bee (Apis melli/era) that was probably what is now commonly called foulb rood. Much later, in the 1 8th century, Louis Pasteur made significant contributions to our knowledge of infectious processes by differentiating pebrine and ftacherie diseases of silkworm (Bombyx mori). Other scientists such as Kirby (87) and Bassi 163

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(11) made important discoveries in insect pathology, and these men, along with Pasteur, are regarded as being among the founders of the fields of infectious disease and pathogenic microbiology. It is not our intent to review the history and scope of insect pathology. The literature, beginning with the 1726 article by de Reaumur (54), which probably was the first published scientific documentation of insect disease; is voluminous. Several texts, particularly those by DeBach (50) and Steinhaus (135), alford comprehensive


coverage of the subject area up to about 1960. Also, Steinhaus (134) wrote a review of microbial insect diseases up to 1956. Since the late 1950s and early 1960s, many papers on insect pathology and microbial control have been published. Many of these articles have been reviewed in the treatises by Burges & Hussey (40), Cheng (43). and Bulla (31), as well as by individual reviews (1, 4, 37,41, 60, 67, 71, 74, 88, 104. 113, 144). In these writings, comprehensive information on the theory, efficacy. safety, and practicability of microbes as insecticides was compiled. The purpose of our review is to present the most meaningful information concerned with the biology of bacterial insect pathogens and to point out certain principles and concepts for research in insect microbiology.

COMPARISON OF BACTERIA TO OTHER MICROBIAL PATHOGENS Because microorganisms are ubiquitous in nature, it is not surprising to find a

variety of them associated with insects. Generally, the kinds of microorganisms

involved with an insect reflect the microflora of the surrounding environment. Various kinds of microbes that exhibit pathogenicity in insects are viruses, myco

plasmas, rickettsias, spirochetes. protozoa. fungi, and bacteria.

Over 300 viruses have been isolated from insects and at least 1 SO have been described. Entomopathogenic viruses comprise those'characterized as DNA viruses (baculovirus, iridovirus, poxvirus, parvovirus) and RNA viruses (cytoplasmic polyhedrosis virus, rhabdovirus, enterovirus, and others), as well as many yet to be classified. Characteristic of most viruses is their high degree of host specificity and their ability to spread rapidly from cell to cell. In n at ure th ey cause spectacula r

epizootics and in the laboratory they can be grown to constant titers in insect tissues. An extreme experimental problem has been posed by the lack of reliable methods to quantify viruses grown in tissue culture. With the recent advent of techniques to cultivate viruses in pure form and to enumerate bona fide plaque-forming units (147), further research is now possible on viral attachment, cellular penetration, replication, and host packaging and specificity. Although mycoplasmalike organisms have been observed to infect insects, their etiologic role in disease and their mode of infection have not been elucidated. In those cases studied, the infection appears to be dual, associated with virus or viruslike particles. It has been hypothesized that the etiological agent for aster yellow that multiplies in a leafhopper is a mycoplasmalike organism (10). Few rickettsias actually cause disease of insects, although a number exist in commensural or symbiotic association with specific insects, notably cockroaches.


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The best known pathogen is the only recognized species of the genus Rickettsiella, Rickettsiella popi/liae (26). Other rickettsias have been isolated from insects and assigned specific names under this genus, but it is doubtful that any such classifica tion is well founded. Information on these microorganisms is so scant that definitive criteria are not available to establish separate species. The fact that many rickettsias are pathogenic to warm-blooded animals has dampened enthusiasm for investigat ing possible use of them as insect control agents. Quite justly, most insect microbi ologists who are working to develop pathogens for use as insecticides have excluded the rickettsias from their list of possibilities even though those organisms considered to be Rickettsiella species exhibit limited virulence for vertebrates. For similar reason s spiroch etes have received little attention, except for the few species found ,

in pathogenic association with mosquitoes. Most protozoa pathogenic to insects are in the class Sporozoa and are micro sporoda. One of the most famous causes pebrine disease of silkworm.

Relatively little fundamental information is available on this group of o rgani sms Two main reasons are that (a) many of the type species are poorly defined and (b) methodology is poor for determining l ife stages. A slow kill rate is characteristic of most protozoan infections; the most serious effect is usually decrease in vigor and life expectancy. Fungi represent the largest and most varied group of entomopathogens. Phycomycetes, ascomycetes, basidiomycetes, and fungi imperfecti all contain mem bers that afHict insects. Generally, their host range is broad, although some are specific for only a few insect species. The host invasion route is normally through the cuticle instead of through the oral cavity. Spores are the infective units, and penetration requires spore germination and chitinolytic activity or injury to the insect. Beauveria bassiana and Metarrhizium anisopliae are two fungi that have been used in the past as control agents, particularly in the USSR. PartIy because of a need for better understanding of disease induction and for more efficient means of artificial cultivation, research on insect-fungal interrelationships is lacking. Cer tainly, there is practical significance to studying such a relationship because the fungi may prove to be among the most efficient weapons for controlling insect infestations; many of them form metabolic and biosynthetic products that exhibit antibiotic activity. In the past 50 years numerous bacteria have been isolated, classified, and demon strated in the laboratory to be pathogenic for various insects. Most of these bacteria are classified in the families Pseudomonadaceae, Enterobacteriaceae, Lactobacilla ceae, Micrococcaceae, and Bacillaceae. Among the known bacterial pathogens is Pseudomonas aeruginosa, which is pathogenic to adult grasshoppers, Melanoplus bivattatus and Camnula pellucida. P. aeruginosa also produces lethal septicemia in the greater wax moth (Galleria mellonella) silkworm, locusts, eastern tent caterpil lar (Malacosoma americanum), cutworms, and hornworm (Manduca spp). P. sep tica is pathogenic to the cockchafer and other scarab beetles. The red-pigmented varieties of Serratia marcescens are the most often reported pathogens of insects because of their distinctive color, which enables ready recognition. However, non chromogenic strains of S. marcescens also are pathogenic (135, 139). Outbreaks of disease caused by S. marcescens are common in laboratory-reared insects and, as .


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in the case of many nonsporeforming bacterial pathogens, the organism is highly pathogenic when inoculated into the insect hemocoel; it is only mildly pathogenic when ingested. The bacterium is frequently found in insectary-reared silkworms. The coliform bacteria Enterobacter aerogenes and Escherichia coli kill silkworms and other ledidoptera larvae. Experiments with E. aerogenes infection of grasshop pers suggest that the disease is caused by a virus and that the bacterium is only a secondary invader (135). The bacterium may be transmitted through insect eggs and, on occasion, causes a morbid process in its host. Other data suggest that the bacterium, commonly present in the grasshopper's alimentary tract, could become pathogenic to the insect under certain environmental conditions. Proteus vulgaris. P. mirabilis, and P. rettgeri are three species of the proteus group incriminated as insect pathogens. However, these organisms probably are not significant. The three bacteria can be artificially induced to infect grasshoppers; pathogenicity is correlated with proteolytic activity. Of the Salmonella-Shigella group of bacteria, Salmonella enteritidis, S. typhi, S. schottmuelleri, and Shigella dysenteriae can be induced to infect larvae of the greater wax moth. The latter organism also causes acute enteritis in bees. Micrococci and streptococci are dubious pathogens and, like E. aerogenes, are probably secondary invaders or chance opportunists. The species nomenclature of these bacteria, like many other insect pathogens, is based erroneously on the insect host from which they were isolated; Bergey's Manual ofDeterminative Bacteriology (26) does not recognize many of these microorganisms listed in insect pathology literature as authentic species. Micrococci and streptococci have been isolated from Melolontha melolontha (a cockchafer), the silkworm, the gypsy moth caterpillar (Porthetria dispar), and the so-called processionary moth caterpillar (Thaumetopoea pityocampa). Streptoccus pyogenes is often isolated from flies; S. faecalis is patho genic for wax moth larvae when injected in moderate doses. Rarely have species of Clostridium been isolated from diseased insects in nature. One reason is improper techniques that disallow preferential growth of anaerobic bacteria. Experimentally, Clostridium novyi and C perfringens are pathogenic for the wax moth (Galleria mellonella). Two sporulating obligate anaerobes have been isolated from diseased larvae of the western tent caterpillar (Malacosoma califor nicum) and the so-called Essex skipper (Thy m elicus lineola) (28), and from diseased pine processionary moths. Bacillus cereus is frequently isolated from diseased Coleoptera, Hymenoptera, and Lepidoptera. The bacillus has been found in the southern armyworm (Spodopt era eridania), American cockroach (Periplaneta americana), (8), Indian meal moth (Plodia interpunctella) (125), codling moth larvae (Laspeyresia pomon ella), spruce budworm (Choristoneura fumiferana), and larch sawfly (Pristiphora erichsonii) (31). Presumably, pathogenicity is due to phospholipase C activity (67). B. thuringi ensis and B. popilliae are crystalliferous sporeformers that are commercially pro duced as insecticides. Crystalliferous refers to those organisms that produce discrete crystalline inclusions within the sporangium in addition to the endospore. Hannay (69) suggested the term parasporal body to describe such an inclusion. Both B.

INSECT PATHOGENS

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thuringiensis and B. popilliae produce crystals; B. lentimorbus, another commer cially produced pathogen, does not. The mode of action of these organisms is discussed in detail later.

SYSTEMATICS AND TAXONOMY


Classification and Identification Bacterial insect pathogens are classified according to Bucher (29) as obligate or nonobligate. Obligate pathogens are characterized by the following criteria: (a) associated only with host in nature, (b) host range generally restricted, and (c) not normally culturable on artificial media. Nonobligate pathogens (a) can occur in nature free of host and (b) are culturable on artificial media. Bacteria included under the category of nonobligate pathogens are heterogeneous and most are species of Enterobacteriaceae and Pseudomonodaceae. [Some were isolated and identified as Achromobacteriaceae according to the seventh edition of Bergey's Manual; see (23)]. However distinct they may be taxonomically, nonobligate pathogens within these families are systematically similar in their ability to proliferate in the hemo lymph of susceptible insects, causing lethal septicemia. Insects ordinarily are resis tant to potential pa'thogens. Although these bacteria have full capacity to kill insects once they have gained access to the heinocoel, they lack the ability to generate the large populations in the gut that would increase chances of penetration. Thus, transmission is rare except under conditions of extreme stress, and the potential of these organisms as agents for biological control is correspondingly limited. By far the largest amount of information reported in this context has dealt with sporeform ing bacteria. The eighth edition of Bergey's Manual of Determinative Bacteriology (26) does not categorize bacterial insect pathogens. Rather, it denotes pathogenicity as a charac teristic Where appropriate. Insects do not harbor special bacteria, although there are obligate pathogens of various species. I n the past, many insect pathologists under standably named bacteria for the insect from which they were isolated, sometimes without regard to pathogenicity. Establishing pathogenicity is a lesser problem, however, than properly classifying the organism within a taxonomic scheme. The taxonomic situation with B. thuringiensis is a classic example of the diver gence between taxonomy and the special needs of workers in a specific field of application for a practical system of determinative classification. The location of B. thuringiensis within the genus Bacillus is based on morphological and physiologi cal characteristics commonly used for the genus. Taxonomy based on this system is exemplified by the treatment of the genus in Bergey's Manual (26) and in the recent monograph of Gordon et al (65). Another monograph by Afrikian (2) con tains much determinative information on the genus, but unfortunately it is entirely in Russian and of limited availability. Gordon et al (65) discounted pathogenicity and the crystalline toxin as bases for speciation because the crystalline inclusion is not invariably present in strains of B. thuringiensis from all culture collections. As a result, these authors consider B. thuringiensis a variety of B. cereus in company


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with B. anthracis and B. mycoides. Similarities between B. cereus and B. thuringien sis include a characteristic exosporium, a common spore antigen, and cross-suscept ibility to some bacteriophages, but there are some biochemical differences-ability to utilize citrate, presence of lecithinase, and acetylmethylcarbinol production although they are variable in one or the other organism, and their utility for species designation can logically be questioned. In contrast, insect pathologists and Bergey's Manual recognize the individuality of B. thuringiensis as a separate species. Their position is based primarily on the presence of the parasporal body (crystalline toxin) and pathogenicity for certain insects. (In nature, these characteristics are closely related because pathogenicity usually is associated with invasion occasioned'by action of the crystal in altering the integrity of the insect gut wall.) In other words, the classification of B. thuringiensis by insect pathologists is based on their interest in strains possessing insecticidal properties. Therefore, the taxonomy relates to biochemical criteria, plus serological analysis of flagellar antigens as developed by de Barjac & Bonnefoi (51,52), coupled with complementary results from electrophoretic analysis of esterases from vegeta tive cells reported by Norris & Burges ( lOS). At present, the species is differentiated into 12 serotypes (Table 1) according to de Barjac & Bonnefoi (52), who have given Table I Established serotypesa of B.

ARS Culture Collection Numberb B-4039 B-4040 B-4041 B-4055 B-4042 B-4043 B-4044 B-4045 B-4056 B-4046 B-4047 B-4057 B-4048 B-4049 B-4050 B-4058 B-4059 B-4060

rhuringiensis H

Variety Name berliner

finitimus alesti

kurstaki s o tta dendrolimus kenya e galleriae canadensis

Antigen Serotype 1 2

3a

3a3b 4a4b 4a4b 4a4c 5a5b

Esterase Type berliner

finitimus alesti NDc

sat ta

dendrolimus

kenyae

galleriae

5aSc

ND entomocidus entomocidus

subtoxicus

6 6 6

aizawai

7

galleriae

entomocidus entomocidus-limassol

marrisoni tolworthi

8 9

entomocidus morrisoni tolworthi

ND

toumanoffi

10 11

toumanoffi

thompsoni

12

thompsoni

darmstadiensis

a Based on classification according to de Barjac & Bonnefoi (51). bARS; Agricultural Research Service, Northern Regional Research Laboratory, US Department of Agriculture, Peoria, Ill. eND; not determined.


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the types varietal status (e.g. B. thuringiensis var. alest i). ! Reports by Bucher et al (30) and Cosenza & Lewis (47) demonstrate the utility of the system in classifying new isolates. Toxicity, including both the crystalline toxin and the /3-exotoxin, was proposed as a criterion for the classification by Heimpel (73), but generally this latter property has been questioned because of the graded response shown in host-patho gen interactions and because of the difficulty in standardizing tests (for a discussion, see 1 1 4). B. popilliae, B. lentimorbus, and B. larvae are likewise considered separate species by Bergey's Manual, although Wyss (157) proposed that the milky disease organisms (D. popi lliae and D. lentimorbus) should be integrated into one species with three varieties: D. popilliae var. popilliae (Krieg), D. popilliae var. lentimorbus (Dutky), and D. popil/iae var. melalonthe (Hurpin). Such a classification was sup ported by Krywienczyk & Luthy (89), who demonstrated a common antigen profil e among these species. In contrast, Hrubant & Rhodes (80) examined them by a different antigen serological system and found a lack of common antigens in the individual strains. In much the same way, the status of other apparently related organisms such as D. pulvifaciens (like B. larvae) and B. euloomarahae (like B. lentimorbus; not mentioned in the eighth edition of Bergey's Manual) is in limbo, according to Gordon et al (65), because they are difficult to grow in artificial media or because too few strains are available for decisive characterization. Generally, then, those aerobic sporeformers that are obligate pathogens, or nearly so, present difficu lties

in specific assignment except on a morpholo gical or perhaps serologi cal basis. A number of diverse bacteria produce extracellular compounds toxic to insects. For examp le, activity against mosquito larvae has been reported for B. sphaericus culture liquors (52). However, until they are chemically characterized, any pos sible value of these compounds in taxonomy is dubious. At present, only the ade nine nucleotide analogue, the /3-exotoxin of B. fhuringiensis, has been defined (21, 56). Probably, as DNA base ratios, DNA hybridization, and computer analysis give rise to "cluster species" or "genetic species," the needs for classification at the applied level will tend to diverge from taxonomic considerations. Accepting the likelihood of fewer species and abandoning phenotypic expressions will generate problems in insect pathology, in part because investigators concerned with the action of the organism on insects work directly with isolates and strains intimately associated with insects. For the most part, such organisms are sporogenic, crystallif

erous, and pathogeni c . Taxonomists, in contrast, must consider long-maintained stock strains far removed from the insect that often lack these properties. Differing viewpoints such as those presented here are not unique to the genus Bacillus, but a resolution must be achieved if the systematics of bacteria is to have basic value . to all aspects of microbiology. lIn the eighth edition of Bergey's Manual of Determinative Bacteriology (26). Bacillus thuringiensis is denoted as a species separate and apart from B. cereus (the genus Bacillus was co mpi led by T. Gibson and R. E. Gordon). Unfortunately, serotype 12 (B. thuringiensis var. thompson;) was not included in the list of subspecific or varietal epi thet s.

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Host-Pathogen Relationships and Specificities

The characteristics of bacteria that contribute to pathogenicity or lack of it are generally unknown. Certainly, inability to proliferate in the gut of many insects is not the result of the high pH encountered, although in some Lepidopteran species susceptibility to B. thuringiensis is based on solubilization of the crystalline toxin under alkaline conditions. Moreover, the pH range of 5.8-7.5 in the gut of many insects is not inimicable to most bacteria. Rather, it appears that the low redox potential is restrictive (-100 to -300 m V are reported). Streptococcus pluton (7-9), which causes European foulbrood in bees that have in their gut an Alcaligenes species (referred to formerly as Achromobacter eurydice), grows anaerobically and produces the disease without entering the hemocoel. The occasional pathogenicity of Serratia marcescens may also be attributable to the ability to grow somewhat better than other enterobacteria under low oxygen tension. In contrast, the hemocoel is relatively oxygen-rich because the aerobic state in an insect is produced by oxygen dissolved directly in the hemolymph fluid from breath ing tubules open to the atmosphere. Thus insect pathogens generally must be at least tolerant of oxygen if they are to grow in the hemolymph. Overall. it appears that a bacterium must be capable of growth under both aerobic and anaerobic conditions if it is to induce disease regularly in insects. According to Bucher (27), another characteristic common to pathogenic bacteria is proteolytic ability; among the bacteria he studied. utilization of carbohydrates (including trehalose), fatty acids, or other components of insect hemolymph was not correlated with pathogenicity. Proteolysis has not been detected in Bacillus popilliae. although this organism grows well in the hemolymph of susceptible insect larvae. Among sporeformers, B. popilliae and B. lentimorbus, together with related but more poorly defined organisms, are obligate pathogens that cause milky disease of susceptible insects. The best-studied representatives can be grown in artificial cul ture. However, their complex requirements for growth, and especially for sporula tion, probably exclude survival in nature outside the host except in the spore stage, and spores are formed in significant quantity only within the insect during disease. Nevertheless, commercially available microbial insecticides contain spores pro duced in artificially infected larvae that spread, producing persistent infections in susceptible insect populations. Thus, they are capable of controlling infestations at economically acceptable levels. In the United States. the Japanese beetle (Popillia japonica) and the European chafer (Amphimallon majalis) are the principal pests attacked; elsewhere, larvae of a number of Scarabaeid beetles that infest grasslands are infected by these and closely related organisms. Likewise, spores of B. larvae germinate in the gut of honey bee larvae and cause American foul brood disease, but they do not germinate in the gut of the adult. Indeed, B. larvae has nearly the same cultural limitations as do the milky disease organisms, the physiology of which is so closely attuned to that of the insect that the organism can be considered an obligate pathogen. E. cereus is often considered a pathogen and strains have been isolated from naturally infected Coleoptera, Hymenoptera, and Lepidoptera. According to Heim pel (73), these strains of B. cereus produce phospholipase in quantities sufficient to


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cause pathological changes in susceptible insect gut, thereby aiding entry of the organisms into the hemocoel. As a result, insects whose gut contents are highly alkaline (such as many of the Lepidoptera) are resistant to B. cereus because phospholipase production is inhibited; impractically high doses of these bacteria are required in field tests. Possibly other facultative pathogens occur among the spore formers that could exert pathogenic effects by producing enzymes damaging to insect gut tissues, so that a septicemia results from penetration by gut flora. How ever, these potential pathogens must have the ability to multiply within the some what restrictive environment of the insect gut al)d to produce products active under the conditions that prevail there. A relatively broad spectrum of action, together with ready growth in artificial culture, have made B. thuringiensis the most widely investigated insect pathogen. Over 130 susceptible species have been found among the orders Hymenoptera, Coleoptera, Diptera. and Orthoptera; many of the susceptible Lepidoptera are among the most important pest insects in the world. The toxic parasporal crystal of B. thuringiensis is dissolved under the alkaline-reducing conditions in the midgut of most lepidopteran larvae, and the solubilized protein is digested by the proteolytic enzyme complex of the insect to release one or more toxic fragments. The molecular mode of action of the toxin is not understood. However, the toxicity of pure crystals from different varieties of B. thuringiensis varies several hundredfold for the same insect, and comparable variation exists in the susceptibility of different insects to the toxin of a single variety. There also is wide variation in toxigenicity among strains of a given variety. Preparations of B. thuringiensis crystals can kill insects in much the same manner as do chemical agents. However, the spores also kill insects, and pure crystal formulations are sometimes less effective than spore-crystal mixtures. Vertebrates and plants are unaffected by the toxin, and persistent infection in a natural insect population rarely occurs. Most varieties of B. thuringiensis were originally isolated from different insects, but they are not separable on the basis of toxicity to a species. Indeed, it has been possible to isolate and select single strains of B. thuringiensis with unusual potency against a variety of important pest insects. Toxicity plus yield-per unit-volume of fermentation media is the basis for selecting the strains that are produced commercially. In contrast, effective strains of B. popilliae and B. lentimorbus are selected on the basis of action as pathogenic control agents; persistence is a primary consideration. Thus, strains of the milky disease organisms are selected for field application for on the basis of ability to grow slowly in the host, allowing larvae to remain alive long enough for a maximum number of spores to be formed (C'J IOto/larva). Rapidly growing, highly "virulent" strains may kill the larvae before appreciable sporulation can occur. In this case, the number of spores produced and liberated into the soil is not sufficient to establish continuing control in the insect population. PHYSIOLOGY

In considering the physiological aspects of bacterial insect pathogens, we have chosen to discuss in some detail the sporeformers that fall within the category of

172


obligate and nonobligate pathogens. We alluded earlier to the fact that the bulk of information concerns sporeformers rather than nonsporeformers. Little is known about the latter group, and few investigations have been made over the past 20 years to develop them as control agents. The sporeformers that have received considerable attention are B. thuringiensis, B. popilliae, B. lentimorbus, and B. larvae.


Growth Characteristics B. popilliae, B. lentimorbus, and B. larvae are obligate pathogens. The first two are milky disease organisms that infect various scarab larvae. . The third, B. larvae, causes American foulbrood in honey bees, and the organism is readily isolated from diseased adults. All three bacteria can be maintained in the vegetative state on various artificial media formulated with yeast extract (112). One such medium (designated MD), used routinely for laboratory cultivation, contains 1.5% yeast extract, 0.6% K2 HP04, and 0.2% glucose in distilled water. A characteristic of the growth patterns of all three species is maximum population in 16-20 hr. However, the stationary growth phase for B. popi/liae and B. lentimorbus is 1-2 hr; it may be as long as 16 hr for B. larvae. Then, after stationary growth is complete, the number of viable cells declines rapidly. Unlike other commonly studied sporeform ers such as B. subtilis. B. cereus T, B. megateriu m and B. licheni/ormis, these species form no appreciable number of spores during the culture cycle. With B. popilliae, the highest number of spores attained has been obtained on a solid medium containing yeast extract plus the ingredients of Mueller-Hinton, trehalose, and phosphate (123, 124). Haynes & Rhodes, however, (70) have reported sporulation of B. popilliae in liquid medium containing activated carbon. The quantity and kind of yeast extract appears to determine the extent of sporulation in artificial culture, though there are other requirements for optimum growth and sporulation. Also, spore inocula free of viable vegetative cells are necessary to maintain sporogenicity because asporogenic substrains arise spontaneously on solid and in liquid media. One such substrain (NRRL B-2309N, ARS Culture Collection, Peoria, Illinois) is also asporogenic in Japanese beetle larvae, but it is lethal to the larvae because vigorous vegetative growth. The growth and sporulation characteris tics of four related strains of B. popilliae are summarized in Table 2. Spores accumulate periodically in colonies of B. popilliae after t hree days of vegetative growth on solid medium. Sporulation occurs on the s urface primari ly in the ring near the periphery, which causes slight changes in colony contour. Varia tions do occur, however. Acquisition of spore resistance to drying and to heat, as well as sporulation itself, occur in a stepwise manner (123). B. thuringiensis is a non obligate pathogen. I t can occur in nature free of host (most isolations, however, have been from diseased insects), and it is easily cultured on artificial medium. In fact, a completely defined synthetic medium has been developed for growing a wide variety of strains (99). Unlike the obligate pathogens discussed, B. thuringiensis sporulates readily in the laboratory. Upon reaching peak population in liquid medium, the vegetative cells transform to spores. The formation of an endospore (the dormant state of B. thuringiensis) is a process of cellular division that includes protoplast formation within the cytoplasm of the cell. The ,

,


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developmental process occurs in an orderly manner and comprises the foHowing stages: (0) axial formation (localization of genetic material along the longitudinal axis), (b) forespore development, which involves invagination of the cytoplasmic membrane and engulfment of genetic material, (c) cortex synthesis and spore coat deposition, and (d) dehydration df spore protoplast with concomitant dipicolinic acid and calcium accumulation. The last stage after completion of sporulation involves acquisition by the spore of total refractility and release of the mature spore from the sporangium. The reader is referred to the treatise by Gould & Hurst (66) for a comprehensive analysis of bacterial sporulation. Nutritional Requirements

B. popilliae, B. lentimorbus, and B. larvae are among the most fastidious of Bacillus species. For example, the milky disease organisms require high concentrations of a wide array of amino acids (48, 1 42). Despite a variety of cultural conditions in liquid semisynthetic media fortified with mixtures of vitamins, coenzymes, and amino acids, vegetative cell populations like those observed in insect larvae cannot be achieved; furthermore, no spores are formed. The most effective of these media (highest cell yield) were formulated with yeast extract; although certain organic nitrogen sources can be substituted, beef extract, for example, is a poor replacement. While devising a chemically defined synthetic m edium for B. popilliae and B. lentimorbus, Sylvester & Costilow (142) found that thiamine is an absolute requirement for growth; biotin, myo-inositol, and niacin are stimulatory; riboflavin inhibits growth, and asparagine the ,B-amide derivative of aspartic acid, plus ten other amino acids are absolutely essential. Furthermore, the pyrimidine barbituric acid that stimulates antibiotic synthesis in certain microorganisms was found to stimulate growth of B. popilliae in semisynthetic medium, but the nutritional func tion of this compound is obscure (49). No synthetic medium has been defined for B. lentimorbus. Just as with the milky disease organisms, what specifically prompts growth and sporulation of B. larvae remains a mystery. Many media have been improvised for vegetative growth, including some supplemented with extracts of larval honeybees ( 1 55), egg yolk ( 1 5 6), carrots (94, 1 4 1 ), turnips (95), chick embryos ( 1 43), and pollen ( 1 29). Moreover, some spores have been formed on solid media containing yeast extract and soluble starch or activated charcoal (59), although the efficiency of sporulation on these media was low. However, all of the media used to cultivate B. larvae are rich in organic compounds, which may mean that exogenous carbon sources repress the enzymes necessary for sporulation, i.e. reduced nicotinamide adenine dinucleotide oxidase, proteolytic enzymes, ribonuclease, and certain tricar boxylic acid cycle enzymes. Repression of these enzymes would reduce energy production and the formation of precursors for macromolecular synthesis, conse quently limiting the rate of sporulation. St. Julian & Bulla (115) found that B. larvae is unable to oxidize glutamic acid, so this factor may account in part for its low capacity to sporulate and for its fastidious nature. Bacillus thuringiensis is another matter. Its nutritional requirements were studied by Nickerson & Bulla (99) in a continuing effort to delineate any mechanisms


� """

Table 2 Growth and sporulation characteristics of four related strains of Bacr7lus popilliae Growth Medium or Conditiona MD or IB agar (a)

Acetate agar (a)

MYPT agar

(j)

NRRL B-2309 Parent Strain Transparent colonies 1-3 mm diam

MD or JB liquid (Q)

o:l

NRRL B-2309M

Derived from B-2309

Derived from B-2309S

Transparent amber

Opaque colonies

colonies 3 mm diam;

diam;

3 mm when (j) 5 mm

No spores

when (j) 5 mm diam No spores

Rough and smooth

Such spores dried and replated yield

Opaque colonies 3 mm

B-23095 0.3% spores

diam < 0.1 % spores

Transparent amber colonies 5 mm diam;

Opaque amber colonies

colonies 2-3 mm diam

Z


Table 2 (Continued) Growth Medium or Condition

NRRL B-2309N

NRRL B-2309

NRRL B-2309S

NRRL B-2309M

Parent Strain

Derived from 8-2309

Derived from 8-2309S

Derived from B-2309M

MYPT liquid (j)

Vegetative growth 8 X 108 viable cells/ml No spores Infective by injection of vegetative cells or spores and by feeding of spores 25% spores, 75% granular cells at 4-5 days, 80-90% spores by 14-21 days

Vegetative growth 2 X 1 09 viable cells/ml; 1.5 X 106 spores/ml under special conditions Infective by injection of vegetative cells or spmes; apparently not infective by feeding; viggrous vegetative growth lethal to larvae Sporulation less than

Vegetative growth same as B-2309M, but has no spores

Performance in larvae

Vegetative growth 1.5 X 109 viable cells/ml; 1.5 X 103 spores/ml Origin of B-2309M Infective by injection of vegetative cells; infectivity by feeding unknown Sporulation less than

Nonmotile; malt extract in sold medium increases hyphal-type growth

Nonmotile

Other

Motile, especially cells from solid media; malt extract increases motility

a MD

B-2309

B-2309

Motility extremely rare

Infective by injection of vegetative cells; vigorous vegetative cell growth lethal to larvae No spores

medium contained 1.5% (Difco) yeast extract, 0.2% glucose, 0.3% K2HP04, and 2% agar. JB medium contained ingredients of MD plus 0.5% tryptone. Acetate agar is MD medium with 0.14% sodium acetate substituted for 0.2% glucose. MYPT medium contained beef infusion, casamino acids, soluble starch (Miieller-Hinton ingredients at 1%), 1% yeast extract, 0.3% K2HP04, and 0.05% trehalose. (a) Autoclaved, (j) fIlter-sterilized. bExtracts of baker's or brewer's yeasts prepared at the Northern Regional Research Center, Peoria, Illinois, by mechanical disruption of commercially grown yeast. =

=

z Vl

Q '"1;1

�

8

!i Vl -.J VI


176

BULLA.

RHODES

&

ST.

JULIAN

regulating sporulation and parasporal crystal formation. In this particular investiga tion, a defined medium was developed that supports abundant growth and sporula tion of a wide variety of B. thuringiensis strains. Other media have been formulated (44,127) that commonly allow only minimum growth of just a few strains and poor sporulation of those strains that do exhibit growth. All 1 2 serotypes listed in Table 1 exhibit an auxotrophic requirement for glutamic acid, aspartic acid, and citric acid when they are grown in basal medium (99). None will grow in minimal glucose-salts media. and the contention that B. thuringiensis grew in glucose-salts media (114. 127, 128) is misleading because the salts used in these media were organic salts that could have accounted for any growth observed. Neither succinate nor fumarate can replace glutamate, aspartate, and citrate, but the requirement for these compounds apparently is neither the result of defects in either glucose catabolism or tricarboxy lic acid cycle activity (101) nor a manifestation of an unfilled vitamin requirement. The requirement for these organic acids can be relieved if basal medium (99) is supplemented with either cystine, thiosulfate, or ethylenediaminetetraacetic acid (EDTA). The physiological function of these diverse compounds is not clear, but citrate and EDTA are positive regulators of fatty acid synthesis (96, Il l , 151, 152) and cystine controls membrane synthesis (146). Perhaps B. thuringiensis requires a stimulator of fatty acid synthesis in a defined medium to grow, sporulate, and form parasporal crystals. Unique Metabolic Properties

B. popilliae, B lentimorbus, B. larvae, and B. thuringiensis all utilize carbohydrates for energy production during vegetative growth. Lactic acid, acetic acid, and CO2 are the primary products of glucose catabolism in the obligate pathogens. In B. thuringiensis, glucose is catabolized completely to CO2, Pepper & Costilow (106) found in B. popilliae and B. lenlimorbus that glucose is metabolized by the Embden Meyerhof-Parnas(EMP) and pentose phosphate(PP) pathways. Participation of the EMP pathway in rapidly dividing cells of B. popilliae is 75%, and in B. lentimorbus 85%; the PP pathway operates at 25% and 15% in each organism, respectively (35). B. popilliae also utilizes trehalose (18); in fact, it catabolizes trehalose (which is the predominant sugar in Japanese beetle hemolymph) more readily than glucose. Fur thermore, the pathway of trehalose utilization differs from that reported for other organisms because the compound undergoes phosphoenolpyruvate-dependent phos phorylation. The trehalose 6-phosphate formed is actively transported across the cell membrane by a phosphoenolpyruvate:sugar phosphotransferase system and is degraded to equimolar amounts of glucose and glucose 6-phosphate. The enzyme responsible for cleaving trehalose is newly identified and has been tentatively named "phosphotrehalose" (I 8). What significance this novel mechanism has on the physi ology and sporogeny of B. popilliae is not yet understood. Three features make B. larvae unique among all other Bacillus species studied: 1 . possession of enzymes peculiar to the Entner-Doudoroff(ED) pathway, 2. glucose catabolism by a predom inantly oxidative scheme, and 3. capacity to metabolize carbohydrates by three pathways operating simultaneously. Whether the latter actually occurs has not been proven experimentally, but radiorespirometric analyses (115) of specifically labeled ..


INSECT PATHOGENS

177

glucose indicate a peculiar pattern of radioactive CO2 evolution that could be explained by concomitant metabolism via the PP, EMP, and ED pathways. In any case, the predominant mechanism of carbohydrate dissimilation appears to be to be the PP pathway, rather than the ED or EMP pathways, operating in conjunction with a tricarboxylic acid cycle. This oxidative activity is further enhanced by exten sive cycling of carbons via pentoses that can be rerouted into PP reactions. Such pentose cycling is not significant in either B. popilliae or B. lentimorbus. The primary metabolic pathways in B. thuringiensis are similar to those in other bacilli in that the EMP is the primary mechanism for glucose assimilation, whereas the PP pathway, by supplying reduced nicotinamide adenine dinucleotide phos phate, aids formation of biosynthetic intermediates rather than functioning as a major respiratory pathway. Tricarboxylic acid cycle activity, commonly associated with capacity to sporulate, is characteristic of B. thuringiensis cells undergoing transition to sporulation ( 160). Although fiuoroacetate, an inhibitor of aconitate hydratase, effectively prevents acetate oxidation, and spore and parasporal crystal formation, it is not certain that this inhibition of tricarboxylic cycle activity abso lutely precludes sporulation. Yousten & Hanson ( 159) demonstrated that the tricar boxylic acid cycle is not an absolute requirement for sporulation by deriving sporulating mutants of B. subtilis with lesions in the cycle. Furthermore, Nickerson et al (1 00) induced sporulation in ce\1s of B. thuringiensis that exhibited no operative tricarboxylic acid cycle. Instead, spores were produced in a medium with extremely low concentration of glutamate and could be described as follows: (a) refractile, ( b) octanol resistant, (e) resistant to 62°C for I hr, and (d) not stained by fat and metachromatic granule-specific stains. The spores were (a) sensitive to heat treat ment (80°C for 30 min), (b) reduced in dipicolinic acid content, and (e) of decreased density. B. popi/liae and B. lentim orbus differ from B. thuringiensis and B. larvae in not h avin g a fully operational tricarboxylic acid cycle. McKay et al (98) found that oligosporogenous mutants of B. popilliae oxidi ze acetate, but they were unable to correlate this acetate-oxidizing capacity with sporulation. These results, along with the fact that tricarboxylic acid cycle enzymes cannot be derepressed in B. popilliae (34) and that B. thuringiensis sporulates in the absence of cyclic activity, lead us to believe that activation of this cycle is not requisite to bacterial sporulation but is

rather only a fortuitous metabolic coincidence. This supposition is further strengthened by recent experimental results of G. St. Julian and L. Bulla (unpub lished) obtained from enzymatic and radiorespirometric analyses of vegetative and sporulating B. popilliae cells harvested directly from diseased third instar Japanese beetIe larvae. The assays of six tricarboxylic acid cycle enzymes support the radiore spirometric data, which indicate that B. popilliae does not possess a fully operational cycle. More importantly, the data show that normal sporulation of this bacterium within the insect host does not necessitate activation of a tricarboxylic acid cycle. To what extent the pathogen may depend on the metabolism of the host is unknown. However, Bulla & St. Julian (32) have demonstrated an active tricarboxylic acid cycle in healthy and diseased Japanese beetle larvae. Also, the PP pathway predomi nates in cells grown within the insect host (only minor involvement of the EMP

1 78


pathway), whereas the EMP pathway predominates in cells cultured in artificial media. The only explanation we can offer for this dramatic shift is that enzymes of the EMP pathway are derepressed by a compound(s) in the insect hemolymph and that depression of certain EMP pathway enzymes occurs when the organism is removed from its host and cultured artificially.


Secondary Metabolism and End-Product Synthesis

As remarked, the most widely recognized and most intensely investigated bacterial insect pathogen is B. thuringiensis. However, the overwhelming majority of research on this organism has dealt with its efficacy in field applications. When prepared as a commercial insecticide, it can be used to protect vegetables, field crops, ornamental plants, and fruit, forest, and shade trees. It effectively controls pest insects such as the cabbage looper ( Trichoplusia nO, imported cabbageworm (Pieris rapae), horn worm, alfalfa caterpillar ( Colias eurytheme), tobacco budworm (Heliothis virescens), green cloverworm (Plathypena scabra), fruittree leaf roller (Archips argyrospilus), orangedog (Papilio cresphontes), grape leaffolder (Desmia !uneralis), cutworm, spruce budworm gypsy moth, and western hemlock looper (Lombdina fiscellaria lugubrosa), to name just a few. How B. thuringiensis kills these insects is still unknown. Death can result from ingestion of (0) the parasporal crystal, (b) both the spore and the crystal, or (c) the spore alone. Mqst economically important lepidopteran insects are killed primarily by ingestion of the spore and crystal or of the crystal itself. Consequently, the parasporal crystal [otherwise known as a-endotoxin after Heimpel; see (72)1 has become a significant bacterial end product. Other toxic agents synthesized by B. thuringiensis are a-exotoxin (the enzyme phospholipase C), {3-exotoxin (thermostable adenine nucleotide), and y-exotoxin (unidentified thermolabile phospholipase). Somerville ( 1 30) and Lecadet (9 1) have reviewed these toxins and presented the most recent information about them. Suffice it to say that the a-endotoxin provides one of the best examples of a biological insecticide. Nevertheless, surprisingly little is known about its structure and molecu lar mode of action. The parasporal inclusion of B. thuringiensis is crystalline (79, 90, 102, 103) and is synthesized within the sporulating cell during stages II and III of sporulation. Presumably, subunits are synthesized and progressively assembled to produce a crystal structure that becomes refractile to light. The component(s) that imparts toxicity is unknown. The first definitive work on formation of the crystal and its relationship to the spore was by Young & Fitz-James ( 1 58), but earlier researchers had shown that the crystal is produced in vegetative cells committed to sporulation (5, 58, 68 , 69) The spore coat protein and the crystal are immunologically and biochemically similar (53, 92, 1 32): both can be readily dissolved in urea-mercaptoethano) or in dilute alkali. Extracts prepared in this manner exhibit the same electrophoretic protein binding pattern in acrylamide gels, and peptide maps of tryptic digests are identical. What physiological function the crystal has in the bacterium is not under stood, but it may be a manifestation of unregulated spore coat protein synthesis. Somerville (J 31) derived some crystalliferous, asporogenic mutants resistant to .

INSECT PATHOGENS

1 79

streptomycin and penicillin, and these mutants produced a substance that was toxic

to fourth instar cabbage worms; the sporogenic. acrystalliferous mutants did not. These results strongly suggest that the I)-endotoxin rather than the spore imparts the entomocidal acti vi ty They also indicate that the spore components, though .

immunologically similar. are not toxic. Lecadet et al (92), however inferred that a .

crystallike, sol uble protein is a componen t of spore coat and could be related to other


structural proteins of the spore. Whether this protein is toxic was not determined.

Earlier work by Lecadet & Dedonder (93) also pointed to a relationship between the soluble crystal protein and spore coat protei ns

.

Detailed biochemical analysis of the S-endotoxin have not yet been accomplished,

primaril y because of the solubility characteristics of the protein and the rather crude analytical techniques used. Cooksey (45) outlined the methods utilized for purifica

tion of crystals and subsequent chemical analyses. The 8 endotoxin is comprised -

nearly entirely of amino acids, and a compilation of amino acid compositions of

crystals from

various

B. thuringiensis strains has been made

(45).

The precise

(76) thuringiensis vaT. tolworthi into two polypeptide molecular weights of 55,000 and 1 20,000; they appeared in a ratio of

number of polypeptide components in the crystal is not known. Herbert et al

separated the crystal protein of B. chains with

I :2. and the component with a mol wt of 55,000 accounted for most of the toxicity of the dissolved crystal. Sayles et al ( 1 1 9) dissociated the crystal into several subunits of low molecular weight ("-' 1000) by using ultracentrifugal analysis. First, it was

dissolved in 8

M

urea

and 0.5%

dithiothreitol (pH 6.0) and then fractionated by

gel chromatography. Although most evidence indicates that several polypeptide components are associated in the crystal the exact number and properties of these .

are not defined. Reported molecular weights of the components range from 1000 to 700,000 (3, 63, 76, 79, 107, 1 19). Most of the techniques utilized to elucidate the toxic moiety of I)-endotoxin have involved drastic conditions such as high pH or elevated temperature or both. Such treatments lead to hydrolysis of peptide and amide bonds that will ultimately result in erroneous molecular weight values for the parent toxin.

Some preliminary chemical characterization of the parasporal crystal of B. popil

liae has been don e

( 1 53).

Crystals were separated from spores by centrifugation in

linear cesi�m chloride gradients and solublized in either 8 M guanidine hydrochlo

ride, 8 M urea, thioglyco\late, or

0. 1 N

sodium hydroxide. The crystals appear to

be predominantly protein and separate into three cathodic components when sub jected to high voltage electrophoresis. No lipid is present. The preparation also contains carbohydrate, however, which suggests the presence of glycoprotein

ponents. If B. popilliae crystals do contain glycoproteins, they

would

com

be the first

reported for a crystaUiferous sporeformer. Indeed, i n our search of the literature.

we found no reference to whether

B. thuringiensis

crystals

contain

nucleoprotein,

lipoprotein, or glycop rotei n components. Of the 17 amino acids present (see Table 3 for com parison with B. thuringiensis). glutamic acid and aspartic acid predomi

nate, followed. in order of quantity, by leucine, valine, glycine, threonine isoleucine, ,

alanine, and arginine. As with the B. thuringiensis crystal (73). no unusual amino

acids were observed. Carbon repli cas of B. popilliae crystals reveal t hem to be

1 80

BULLA, RHODES

&

ST. JULIAN

Table 3 Amino acid analyses of B. popilliae and B. thuringiensis parasporal crystals (grams of amino acid residues per 1 00 g) Organism

B. popilliae

Tryptophan


B. rhuringiensis b var. berliner

NRRL B-2309 a

Amino acid

NOC

5.2

Lysine

2.95

4.0

Histidine

1.10

Ammonia

NO

2.5 2.0

Arginine

4.5 1

8.5

Aspartic acid

7.48

Threonine

3.84

1 1 .9 5.2

Serine Glutamic acid

2.78

5 .4

1 3.56

1 2.5

Proline

0.0

4.0

1/2 cystine

0.0

NR

Cystine

NRd

Glycine

0.3 3.0 :U 5.7

2.28

Alanine

2.06

Valine"

4.06

Methionine

0.66

1 .6

I soleucine

3 .40

6.3

Leucine

4.75

8.8

Tyrosine

2.7 7

5.9

2.65

5.2

Phe nylalanine a A R S C u ltu re Collection, Peori a Ill.

b Ho imes & Monro (79). C

NO

=

not determined.

d NR

=

not reported.

,

bipyramidal, with a distinctive subunit structure; the average distance from ridge to ridge on the crystal surface is 0.Ql7 !-lm . No X-ray diffraction data are available, nor have any electron micrographs been made of sections through isolated crystals. Black ( 1 9, 20) has investigated the morphologi cal development of spore and crystal

within the bacterial cell. B. thuringiensis produces an extracellular, heat-stable toxin (l3 ex otoxin ) that is toxic to a variety of insect species (42). The structure of l3-exotoxin elucidated by Farkas et al (56) and Bond et al (2 1 ) contains an adenosine derivative linked through a glucose moiety to the 5'-position of phosphoallaric acid. The molecular weight is about 700 (56, 1 2 1 ) and the toxin is a competitive inhibitor of RNA polymerase of E. coli and mammalian cells ( 1 2, 1 20-1 22). The l3-exotoxin, originally referred to as "fly factor" (22) because of its toxicity to the house fly (Musca domestica), is also produced by B. cereus (97). The role of l3-exotoxin in the metabolism of B. thuringiensis appears to be that of a posi t ive modifier of RNA polymerase, and it may also function during sporulation. Johnson et al (84) examined an in vitro system of B. thuringiensis var. berliner and found that a concentration of 1. 2 5 X 1 0""4 M J3-exotoxin caused a 50% inhibition of vegetative RNA polymerase activity, -


INSECT PATHOGENS

181

whereas it effected only a 30% inhibition of sporulation-specific RNA polymerase. The differential inhibition of transcription rates of the two enzymes in the presence of J1-exotoxin was apparent with B. subtilis or calf thymus DNA as template, but no variation in transcription rates was evident with a synthetic template such as polydeoxyadenylic-thymidylic acid. In other work related to regulation of RNA polymerase in B. thuringiensis. Iandolo & Bulla (82) found that dormant spores contain an inhibitor of vegetative cell polymerase, and that inhibition of transcription is relieved very early during spore outgrowth. Release of the inhibitor also can be accomplished by addition of boiled vegetative extracts, which indicates that a heat-stable compound (probably a small molecule) is present in vegetative cells. Such a mechanism for regulation of transcription is consistent with the kind of inhibition of the sigma subunit in B. subtilis proposed by Tjian & Losick (145). PATHOLOGY Basis 0/ Pathogenicity

What precisely is a bacterial insect pathogen? According to Bucher (29), it is "a �icroorganism that is capable of growing, multiplying, or developing within or upon a host organism and that by so doing causes the individual host some demon strable harm." From this definition, it can be realized that insects become diseased without outwardly exhibiting any abnormal symptoms. J ust as other animals over come bacterial infections, so do insects contract disease, become sick, and then recover to carry on apparently normal activities. Most information about bacterial insect infections stems from studies of lethal diseases of economically important pests; much less is known about the microorganisms that inhabit other insects in nature. Insect disease may be enzootic or epizootic. An enzootic disease in a population may go unnoticed because so few insects exhibit a particular syndrome. Enzootic diseases generally are always present and become distinguishable only when they reach outbreak proportions (epizootic). In nature, insect disease fluctuates from enzootic to epizootic depending greatly upon environmental factors. Conditions that favor a microbial pathogen can enhance the disease to an epizootic state and those that are adverse to the pathogen suppress it to an enzootic state. Once a bacterium has been recognized in association with a disease state, it is necessary to establish its identity as a disease agent separate and apart from other organisms that comprise the host microflora. In other words, it must meet all criteria of Koch's postulates: 1 . the organism is always found in infected hosts but is not present in healthy ones; 2. the organism can be grown in pure culture outside the host; 3. pure cultures, when inoculated into susceptible insects, initiate the charac teristic disease symptom(s); and 4. the organism can be reisolated from the experi mental insect and recultured artifically, retaining the characteristics of the originally isolated organism. In the case of nonlethal infections, recognition of the disease syndrome(s) is imperative for proper diagnosis, but again, according to Bucher (29),

1 82

BULLA,

RHODES & ST.

JULIAN

establishing pathogenicity and indicting an incriminated microbe depends on the amount of appropriate information. We believe, as Bucher suggests, that a probit regression would provide the most reliable evidence in determining pathogenicity (Koch's postulate number

3). Basically, such an exercise includes these steps: (a)

administration of a graded series of doses of the agent to a selected number of test insects; (b) measurement of the effect in terms of the percentage developing disease;

(c)

conversion of percentages to probits; and

(d) plotting of the probits against

log


dose. If the disease occurs randomly with no true regression, the microbe in question is probably not the etiologic agent. If, however, the occurrence of disease is high with low dosages of the microbe, pathogenicity may be ascribed to it. It is not always so easy to ascertain etiologic relationships because sometimes the results are partially negative. Nevertheless, to conclude that a bacterium is pathogenic on circumstantial evidence is to base proof on inference rather than on scientific process. Conse quently, it is important to adhere to the canons of Koch. Heretofore, many micro organisms have been touted as pathogenic when in actuality they were only asso ciated by happenstance with a disease state.

Invasion Routes The primary route of entry by bacteria is the oral cavity, although there are reports

( 1 36) of bacterial pathogens that attack the outer integument. Insect integument consists of the cuticle (outer body wall) and the/epidermis (inner body wall). Little information is available on bacteria that penetrate the integument despite the fact that a considerable number of them are reported to break down chitin, the chief component of insect cuticle. The genus

Beneckea

contains the largest number of

microorganisms reported to degrade chitin. Bacteria also are secondary invaders of insects through wounds caused by fungi, nematodes, mechanical injury, and para sitic or predacious insects. In the latter case, pathogens can be inoculated into healthy insects by contaminated stingers or ovipositors of the preying insects; thus, predator and parasite insects can facilitate multiplication, distribution, and trans mission of pathogens without themselves becoming diseased. Once a pathogen has gained access to the insect via the oral cavity, several factors must be overcome before it can establish itself as a threat. Conditions in the gut that restrict bacterial proliferation are high hydrogen ion concentration, low oxidation reduction potential, and antibacterial substances in the insect food (mostly of plant origin) (75). If a pathogen can establish itself in the insect gut, it may cause sickness or starvation to the host simply by reducing the amount of nutrients. However, such a disease condition is unstable and the microorganism, to be successful, must further fortify itself within the host. The pathogen establishes itself by penetrating the epithelial lining of the gut and invading the hemocoel containing the blood. Here again, however, it is met with other defense mechanisms, including humoral and

-� _______cellular

responses. Although specific humoral and cellular defense mechanisms like

those of vertebrates have not been defined, we presume that phagocytes and antibac terial substances contained in the blood help aid the insect to fight off invading bacteria. Several reviews have been written on this subject

(24, 75, 1 39, 1 50);

the

reader is referred to these articles for a more detailed analysis. Bacteria that break down all of these defense barriers can eventually kill the insect.

I NSECT PATHOGENS

1 83


Host-Pathogen Interactions

The reader will have noted that no molecular interaction of any bacterial pathogen with its insect host has been elucidated, not even for B. thuringiensis and B. popilliae, the most thoroughly studied examples. The general effect of the parasporal crystal of B. thuringiensis on lepidopteran larvae was reviewed in 1 960 by Heimpel & Angus (74). Since then, little work has been reported to resolve the mode of action by the crystal in molecular terms. Events attributed to parasporal crystal activity are: (a) gut paralysis and sometimes general paralysis of the body; (b) enhanced secretory activity of gut epithelial cells ( 1 54); (c) separation of gut cells and detach ment from the basement membrane (25); (d) increased permeability of the gut wall to sodium ions with a slower rate of glucose uptake into the hemolymph (57); (e) rise of potassium ion concentration in the hemolymph ( 109); and (j) blockage of nerve conduction (46). Other studies of the insecticidal activity of B. thuringiensis has dealt with the heat-stable /1-exotoxin. As already discussed, the compound is competitive with adenosine triphosphate (ATP) for the active site on RNA polymerase ( 1 20, 1 2 l). Whether the compound preferentially inhibits RNA polymerase in insects is not known, but other metabolic functions such as synthesis of DNA and protein in mice are unaffected by doses well above the mean lethal dose (LD5o) value. It stands to reason that any ATP analogue such as the /1-exotoxin could inhibit other enzymati cally catalyzed reactions rather than be selective for only one. Rigorous examination of its effect on other physiological functions is required before any firm conclusions can be made. The most comprehensive investigations of the biochemical interaction of a bac terium with its insect host are those by Bennett & Shotwell ( 1 6). These investigators examined many aspects of the milky disease process in Japanese beetle larvae, by measuring organic acids ( 1 40) and individual amino acids ( 1 25, 1 26), characterizing hemolymph proteins ( 1 4, 1 7), and analyzing lipids and hydrocarbons ( 1 3, 1 5). Concentrations of organic acids such as malic, glycolyic. tartaric. pyruvic. and glyoxylic acids were higher in healthy than i n diseased larvae. Concentrations of butyric, propionic, acetic, formic, succinic, lactic, citric, and a-ketoglutaric acid concentrations, however, were the same. Gluconic and oxaloacetic acids were present in trace amounts and have not been quantitatively determined. Large amounts of several free amino acids were present in larval hemolymph: glutamine, arginine, histidine. proline. glycine. alanine. valine. and cystine. Others occurring in detectable amounts included glutamic acid, aspartic acid, lysine, serine, threo nine, leucine, isoleucine, tyrosine, phenylalanine, tryptophan, asparagine, lanthio nine, ornithine, and l3-alanine. The total amount of amino acids did not vary from healthy to diseased larvae, but there were fluctuations in individual acids. Glutamic acid, /1-alanine, aspartic acid, phenylalanine, threonine, serine, and lysine were more abundant in diseased than in healthy hemolymph; conversely, glycine. histi dine, and tyrosine were reduced. No differences occurred in the amounts of trypto phan, cystine. cysteine, glutamine, asparagine, and lanthionine. The major protein was a lipoglycoprotein whose concentration decreased drasti cally during the infectious process. Fatty acids of significant quantity contained in


I S4


the protein were palmitic, stearic, oleic, linoleic, and lignoceric acids. Identified individual lipid components were diglycerides, sterol esters, methyl esters, triglycer ides, and sterols. The carbohydrates present were mannose, glucose, and galactose. Trehalose, the predominant sugar in larval hemolymph, was not present in the lipoglycoprotein. The total.amounts of lipids and hydrocarbons decreased during milky disease, but the decrease in lipids was nonselective for neutral lipids and phospholipids. The neutral lipid fraction was composed of sterols, monoglycerides, free fatty acids, 1 ,2and 1 ,3-diglycerides, triglycerides, sterol esters, and hydrocarbons. The phospholip ids consisted essentially of phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid. Predominant fatty acids of both the neutral lipids and phos pholipids (in decreasing concentration) were as follows: CIS'" C1S,2' CI6,O, CIS,o, C 1 6,1> CI SJ , or C20,o, and Cn,I' The major hemolymph hydrocarbons were 9, 1 3-dimethyltricosane (27%), 1 1 methyltricosane ( 1 9%), tricosane ( 1 2 %), and I l m et hy l pen tacosa ne ( 1 1 %). Al -

though the overal l concentration of the hydrocarbons decreased during developing

disease, the relative concentrations of the individual hydrocarbons remained con stant. Thus, it is unlikely that B. popilliae, the disease agent, utilized the hydrocar bons for biosynthetic purposes; rather, it simply interfered with hydrocarbon synthesis. There is no conclusive explanation of the cause(s) of larval death from milky disease. No toxic compound(s) excreted by the bacterial pathogens have been identi fied; physiological starvation of larvae during the infection, as suggested by Bulla & St. Julian (33), has not been proved; and conditions within the living larvae that allow for abundant sporulation ( 1 17, l I S) have not been elucidated.

USE OF BACTERIA AS INSECTICIDES Basic Requirements

Generally, the princi ples that govern successful control of insect pests by chemicals The prime factor, of course, is that the microbial agent must be active against the target species without affecting man, other animals, and plants. Some of the features desirable for microbial control agents have been identified (39); the agent should be (a) conveniently applicable (could be applied as dust, spray, or bait and could be used in conjunction with chemicals, predators, parasites, other pathogens, or various combinations of these), ( b) storable, ( c) economical, ( d) easily producible, (e) virulent, and (j) safe and aesthetically acceptable. Application should be made at the time the target insect begins to feed and prior to any major economic damage to the commodity. A lethal or pathogenic quantity must necessarily reach the insect in a vehicle suitable for ingestion by the insect, and this vehicle should not be detrimental to the pathogen. No harmful residues should remain on the commodity treated, especially when diluents or carrier materials are used. Whether a microbial insecticide is successful depends upon many things. The defense mechanisms of the host are of primary concern, but several environmental

are adequate guidelines for the use of bacterial insecticides.


INSECT PATHOGENS

1 85

and physical factors are also important. For example, sunlight, temperature, rain, relative humidity, mode of application of the agent, the vehicle used for it, and standardization of application determine the effectiveness of short-term and long term insect control. Spores of B. thuringiensis and B. popilliae are able to survive most environmental changes that occur in the habitats of their insect hosts, whether they are applied to field crops or to commodities held in storage. Various techniques have been employed to protect microbial agents with greater attention to viruses than to bacteria. Raun & Jackson ( 1 1 0) have described an encapsulation method for the preparation of both microbial and chemical insecticides. Bacteria are capable of infecting insects only by avoiding or overpowering the defense mechanism(s) of the insect host. If the fundamental aspects of a particular host-pathogen relationship are understood, control by a pathogen can be made more successful for a particular insect. Also, any exercise in microbial control must include proper scheduling of application of the pathogen as well as selecting the most desirable vehicle consisting of materials that enhance efficacy. A comprehen sive analysis of this subject has been presented (55).

Experimental and Field Applications Extensive field testing of the efficacy of B. thuringiensis, B. popilliae, and B. lentimor bus has been done for a long time. Preparations containing these bacteria are now commercially produced and are used to control a large number of insect pests. These insecticides do not affect the biocenosis; they pose no threat to man and animals and do not pollute the environment. Furthermore, no changes in toxicity or virulence for their insect hosts have been reported, and the insects have developed no apparent resistance to the microorganisms. Unfortunately, other bacteria that might also be used as control agents have not been examined as thoroughly as have these three organisms. The commercial preparations of B. popilliae and B. lentimorbus spores are ob tained from diseased Japanese beetle larvae and are applied as dust to soil infested with Japanese beetles or European chafers. These insects collectively feed on more than 200 species of plants and, if not controlled, they annually destroy crops worth millions of dollars ( 1 1 6). B. thuringiensis has now been field tested in integrated control and pest manage ment systems (8 1 , 144). This organism, combined with nuclear polyhedrosis virus, increased the control of the alfalfa caterpillar and the western tent caterpillar ( 1 33, 1 37, 1 3 8). In combination with the fungus Beauveria bassiana, it has provided effective control of the so-called European cabbageworm (85). Reviews by Pristavko ( 1 08), Vankova ( 1 49), and Herfs (77) point out that B. thuringiensis is compatible with a wide range of fungicides, insecticides, herbicides, and acaricides. In addition, Pristavko ( 1 08) discussed the probable increase in the effectiveness of one or both of the combined insecticidal agents, and the effect of this combination on dose reduction. Bacteria have also shown promise in the protection of stored products and control of fowl body lice: B. thuringiensis controls several pests of stored flour, corn meal, almonds, and tobacco (36, 38, 62, 64, 86, 148, 1 6 1). Hoffman & Gingrich (78) found


186


that dusting chickens with B. thuringiensis spores and crystals effectively reduced louse populations and did not harm the animals. Interestingly, the major large-scale field testing of B. thuringiensis has been done in Europe and the USSR. Franz & Krieg (6 1 ) reported success in regulating popula tions of the so-called Siberian silkworm (Dendrolimus sibericus) in the USSR, the oak tortrix ( Tortrix viridana) in the USSR and Europe, and the grey larch tortrix (Zeirophero diniono) in Europe. Indeed, the USSR has the most comprehensive program, compared to that of the United States and Europe. At the annual meeting of the Society of Invertebrate Pathology in June 1974, Dr. W. Klassen of the ARS, Beltsville, Maryland, reported on the microbial control program in the Soviet Union. The goal of the USSR is to have 70% of its microbial control agents developed by 1 980. Currently registered products in the USSR are (0) Entobactrin3, which contains B. thuringiensis serotype 5 and is .claimed to be virtually free of exotoxin-it is effective against 85 species of lepidoptera and is used principally on orchards, cabbage plants, shade trees, and forests; ( b ) Dendrobacillin (serotypes 4a, 4b), which contains small amounts of p-exotoxin-itis applied by spraying or in bait form and is used to control Dendrolimus sibiricus on forests, and Laphygina oxiguo, Argrotis segetum. and Chloridea obsoleta on cotton; ( c) Boverin (Beauverin) which. combined with Entobacterin-3, can be impregnated in bands around tree trunks to serve as centers of infection-it is based on highly virulent strains of the fungus Beauveria bassiana and is registered for use against the Colorado potato beetIe (Leptiizotarsa decleminota). the codling moth, and the oriental fruit moth (Grapholi. tha molesto). Safety and Environmental Implications

The overriding concern in using bacterial pathogens to control insects is their safety to man and vertebrates, although the risk of infecting man with an invertebrate bacterial pathogen is probably very low. However, scientists working with such organisms have an obligation to assess such risks. Tests designed for such purposes include examinations of (a) acute and chronic toxicity and pathogenicity for ani mals, (b) primary irritation to eye or skin, (c) teratogenicity and carcinogenicity, (d) selection of mutants for possible pathogenicity, (e) specificity of pathogenicity for invertebrates, (j) potential pathogenicity for plants, and (g) in vitro assessments of cytopathic effects, hemadsorption, and interference-this last evaluation being more appropriate for viruses (83). B. thuringiensis and B. popil/iae have both been SUbjected to most of these tests and demonstrated to be virtually harmless to vertebrates and other nontarget pests. Ignoffo (83) has outlined guidelines for studies on the safety of entomopathogens for vertebrates, and we believe certain them to be important and relevant to the development of any microbial insecticide: (0) the presumed presence or absence of safety based on comparative analysis of closely related species should be confirmed by rigorous evaluation of the specific entomopathogen in question; (b) safety for vertebrates is relative and points to consider in evaluating data are the dose and the manner in which the agent was administered; and ( c) toxicity or pathogenicity can be demonstrated when no dosage limitations are ,t and when no restrictions are placed on the kind of vertebrate system tested.


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It is much too early to assess adequately the environmental impact that insect control by microorganisms will have in the future. However, biomagnification of chemical pesticides is a problem that must be overcome. Presumably, safer alterna tives will include bacterial agents. Few data have been reported on.persistence, and none on concentration, of microbial agents in the food chain. Certainly, the current commercial microbial insecticides are harmless to vertebrates and other nontarget life forms. What is urgently needed are firm scientific data to establish criteria that delimit environmental factors for specific geographical areas. Fundamental studies of the ecology of insects and their pathogens is required to gain such information.

CONCLUSIONS Interest in bacterial insect pathogens is primarily related to the utilization of the organisms to control pest insects. However, there are other theoretical implications that are important to consider. Particularly, invertebrates possess agglutinins in their hemolymph that can clump a variety of animal cells and that exhibit specificity for human erythrocyte blood groups. Although these agglutinins are probably not produced by mechanisms similar to those concerned in antibody production, both have in common a defense function. In invertebrates, including insects, these sub stances are responsible for parasite immobilization as well as for antiviral and bacteriocidal activity. Most of the experimental questions asked so far are very general. It is hoped that with the advent of new insect tissue culture systems and cell lines, bacterial insect pathogens will be used to gain more basic knowledge of animal defense mechanisms. Man associates with insects and insect diseases from time to time. Insects are important vectors of human disease and the pathogens concerned do not generally infect the insect. The fact that insects harbor bacteria that infect man and other animals raises intriguing questions concerning insect immunity to such pathogens. More intensive investigations on the comparative aspects of insect and vertebrate immune responses would shed considerable light on the phylogenetic evolution of disease. Certainly, information acquired from such studies would enhance our knowledge of pathogens and diseases while permitting a greater understanding of their use in controlling economically important insects and a keener appreciation of how to suppress diseases of beneficial insects and other invertebrates. Literature Cited

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Intestinal regeneration as an insect resistance mechanism to entomopathogenic bacteria.

Commensal bacteria mediated defenses against pathogens.

Pathogens and insect herbivores drive rainforest plant diversity and composition.

Soil bacteria as sources of virulence signal providers promoting plant infection by Phytophthora pathogens.

Viruses versus bacteria-novel approaches to phage therapy as a tool against multidrug-resistant pathogens.

Clostridia as intestinal pathogens.

Staphylococci as urinary pathogens.

Insect Antimicrobial Peptide Complexes Prevent Resistance Development in Bacteria.

Screening of endophytic bacteria against fungal plant pathogens.

The Latex Protein MLX56 from Mulberry (Morus multicaulis) Protects Plants against Insect Pests and Pathogens.

Pathogens as biological weapons of invasive species.

Bacteriophages as pathogens and immune modulators?

Engineered bacteria as therapeutic agents.

Insect-specific production of new GameXPeptides in photorhabdus luminescens TTO1, widespread natural products in entomopathogenic bacteria.

Optimization of recombinant bacteria expressing dsRNA to enhance insecticidal activity against a lepidopteran insect, Spodoptera exigua.

Insect-bacteria parallel evolution in multiple-co-obligate-aphid association: a case in Lachninae (Hemiptera: Aphididae).

Diversity and abundance of phyllosphere bacteria are linked to insect herbivory.

A comparative analysis of recombinant protein expression in different biofactories: bacteria, insect cells and plant systems.

Acetic acid bacteria genomes reveal functional traits for adaptation to life in insect guts.

Fighting Off Wound Pathogens in Horses with Honeybee Lactic Acid Bacteria.

N-acyl-homoserine lactones-producing bacteria protect plants against plant and human pathogens.

Strain-specific probiotic properties of lactic acid bacteria and their interference with human intestinal pathogens invasion.

Inhibition of Fungal Pathogens across Genotypes and Temperatures by Amphibian Skin Bacteria.

Inhibition of food-borne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat.