BACTERIOLOGICAL REVIEWS, Sept., 1965 Vol. 29, No. 3 Copyright (©) 1965 American Society for Microbiology Printed in U.S.A. Symposium on Microbial Insecticides 1. Bacterial Pathogens of Insects as Microbial Insecticides' T. A. ANGUS Insect Pathology Research Institute, Department of Forestry, Sault Ste. Marie, Ontario, Canada INTRODUCTION.................................................................. 364 Classification of Entomogenous Bacteria ................................... 364 Attributes of an Ideal Microbial Insecticide ................................... 364 NONSPORULATING BACTERIAL PATHOGENS 365 Pseudomonas aeruginosa ................................... 365 Serratia marcescens ................................... 365 SPOREFORMING BACTERIAL PATHOGENS ................................... 365 Clostridium Species ................................... 365 Bacillus Species ................................... 366 B. cereus................................................................... 366 B. thuringiensis ................................... 366 LITERATURE CITED ................................... 369 INTRODUCTION associated with a specific disease of specific in- In a symposium on microbial insecticides, it is sects. appropriate to recall that it was Agostino Bassi Applying these criteria, there are many bac- who, in 1838, first proposed that microorganisms terial species that can be classed as insect patho- might be used to control insects (67). About 100 gens but only a few of these have shown any years ago, Pasteur noted the presence of bacteria promise as microbial insecticides; this has been in diseased silkworms, and since that time a large discussed in a number of papers (3, 12, 26, 29, 30, number of associations between insects and 32, 33, 53, 67, 73, 81, 82). The factors that bacteria have been reported (65, 66, 68, 72). govern whether a pathogen is endemic or epidemic in a given population are exceedingly complex, Classification of Entomogenous Bacteria and insofar as insects are concerned our knowl- is at best Bucher (16) proposed a useful classification of edge fragmentary (83). the entomogenous bacteria which has been widely Attributes of an Ideal Microbial Insecticide adopted. In it, obligate pathogens are defined as those found associated with a specific insect Previous attempts to utilize bacteria as micro- disease; they have a narrow host range, are bial insecticides have revealed certain attributes readily transmitted, require specialized condi- that materially affect successful use. First of all, tions for growth, and in nature probably multiply the prospective pathogen should be virulent, at only within the bodies of certain species of least to the extent that it consistently causes a insects. The facultative pathogens are a group of disease serious enough to inhibit the competitive bacteria that possess some mechanism for damag- activity of the pest insect. Variations in virulence, ing or invading a susceptible tissue but are not when they occur, should not be so great as to obligate parasites; they are readily cultured in affect materially a recommended dosage or artificial media and are capable of multiplying require frequent assays. The pathogen should not within the gut of the host insect before invasion be markedly sensitive to the environmental of the hemocoele. Potential pathogens are those hazards to which it will be exposed (such as that normally do not multiply in the gut but can desiccation and sunlight), to the way in which it do so in the hemocoele once they gain access to it; will be introduced (such as a spray or a dust), or they grow readily on artificial media, and are not to the suspending medium (oil, water, stickers, emulsifying agents) used. It should also be I A contribution to the symposium "Microbial persistent, in the sense that it will remain viable Insecticides" presented at the Annual Meeting of the American Society for Microbiology, Washing- or infectious until it gains access to the target ton, D.C., 7 May 1964, under the sponsorship of the insect. In general terms, it is preferable that the Division of Agricultural and Industrial Bacteriol- pathogen be rapid in its action, for it is the feeding ogy, with Harlow H. Hall as convener and Con- activity of most agricultural and forest insects sultant Editor. Paper no. 68 of the Insect Pathol- that makes them pest species; this is not a rigor- ogy Research Institute. ous requirement however. It is important that 364 VOL. 29, 1965 BACTERIAL PATHOGENS AS INSECTICIDES 3s65 the pathogen be fairly specific for the insect pest known whether this toxin plays a role in a natural it is used against, and inactive against the host infection. P. aeruginosa also produces a phos- plant and useful insect species such as parasites pholipase (60); an enzyme of this type is known and pollinators. It is of extreme importance that to be an important toxin in certain entomogenous the pathogen be harmless, under the conditions strains of Bacillus cereus (37). of use, for vertebrates, and especially for mam- mals. Finally, it must be possible to produce the Serratia marcescens pathogen in quantity at an economically accept- S. marcescens Bizio is not nutritionally fastidi- able cost in a form that is practicable and ous, grows in a wide pH range, is a facultative aesthestically satisfactory. Taken as a whole, anaerobe, and is strongly proteolytic; therefore, this is a rigorous set of requirements that excludes one would expect it to be capable of multiplying most isolates from consideration (3, 11, 12, 26, 32, in the gut of many insect species. Although most 33, 53, 71, 83). strains are pathogenic if injected into the hemo- The known bacterial pathogens of insects are coele of insects, only a few cause disease if taken found principally in two families: the Entero- by mouth (18, 39, 58, 69). S. marcescens is often bacteriaceae and the Bacillaceae; a few species isolated from diseased and dead insects; Bucher occur in the Pseudomonadales (18, 45). The first classified it as a facultative pathogen. It produces of the bacteria to be considered for microbial con- a phospholipase (4, 28, 48, 54). Although it is trol was identified by d'Herelle as Coccobacillus recorded as being frequently associated with acridiorum. Early optimistic claims for its useful- natural populations of grasshoppers and locusts, ness in reducing grasshopper populations have and some 75 insect hosts are known, it is more not been substantiated by subsequent attempts commonly isolated from insects reared under to use it. Bucher (14) reviewed all of this earlier laboratory conditions than from insects taken work, and concluded that C. acridiorum is identi- from the field. Red and nonchromogenic strains fied as Cloaca type A of the family Entero- have been isolated (79, 80, 83). bacteriaceae, strains of which are wide-spread The use of such nonsporulating bacterial inhabitants of the gut of grasshoppers. In pathogens of insects is beset with some difficulties, Bucher's opinion, it should not be classed as a among which are greater sensitivity to drying and true pathogen. sunlight, and a greater tendency to variability of virulence. Pseudomonas spp. and Serratia spp. NONSPORULATING BACTERIAL PATHOGENS also have the disadvantage that they include Pseudomonas aeruginosa strains demonstrating some degree of pathogenic- It has been shown that P. aeruginosa ity for mammals (5, 9, 10, 84). However, the (Schroeter) Migula also is pathogenic for the nonsporulating bacteria are responsible for much grasshoppers Melanoplus bivittatus (Say) and mortality under natural conditions, and it is Bucher possible that as we add to our knowledge we will Camnula pellucida (Scudder). classified be enabled to circumvent some of their short- P. aeruginosa as a potential pathogen, that is, a comings. Research is to species which does not normally multiply in the required achieve a better can do so in the understanding of the metabolism, in insects, of insect gut but hemocoele from such types of bacteria, and particularly of their small inocula. Ruptures often occur in the grass- and hopper gut, and it may be that P. aeruginosa oxygen requirements the kinds of proteolytic gains access to the insect hemocoele in this enzymes they produce. As Bucher pointed out, rather than as the result of the relatively anaerobic conditions of the insect accidental way some inhibit or limit of invasive mechanism. This species often causes gut may growth such bacteria in of but and, thus, the subsequent production of lytic disease laboratory rearings grasshoppers, that lead to successful natural infection in field populations has never enzymes invasion of the been demonstrated (15, 18, 19, 20, 77). It is hemocoele (16). known that P. aeruginosa is readily killed by SPOREFORMING BACTERIAL PATHOGENS drying and sunlight. This can be partially offset The sporeforming bacteria of the family by use of a mixture of mucin, sucrose, and casein Bacillaceae comprise the greater part of the as a coating. A limited field trial of P. aeruginosa known bacterial pathogens that have been so protected, as a spray and in baits, failed to seriously considered for use as microbial insec- exert any useful level of control (76, 78). ticides. Lysenko recently demonstrated in P. aeru- ginosa cultures the occurrence of an antigenic Clostridium Species compound that is toxic for larvae of Galleria Species of the genus Clostridium have been mellonella L. by injection (49, 50, 51, 52); it is not isolated only rarely from diseased insects. It 366 ANGUS