Bacterial Diseases
- General Features and Taxonomic Status
- Family Bacillaceae - Bacillus thuringiensis
- Non-sporeforming Bacteria - Serratia marcescens
- Rearing Bacteria-free Heliothines
- References
Bacteria were discovered by Anton van Leuwenhock in 1632. Relatively few species of bacteria cause disease in humans or other organisms, and in fact, without the activities of many kinds of bacteria, all life on Earth would cease. Presently there are 4,000 species of bacteria described, and the estimated number remaining to be discovered ranges from 400,000 to 3 million (McDonald, 1994).
Bacteria have three general morphological types: 1) the spherical form, or coccus, 2) the rod-shaped form, or bacillus, and 3) the spiral forms, the various subtypes of vibrio, sprilillum, and spirochete (may be regarded as bacilli twisted into a helix). Bacteria are the most common microorganisms associated with insects. Generalized symptoms of insects associated with bacterial infections are loss of appetite, diarrhea (discharge of watery feces), and vomiting (Tanada and Kaya, 1993). The invasion of the bacteria into the hemocoel results in septicemia and death of the insect.
Bacteria are extracellular pathogens except for the pathogenic rickettsia and mollicules (mycoplasma and spiroplasma). Insects killed by bacteria usually darken in color and are often soft, but the integument remains intact. The internal tissues and organs are decomposed to a viscid constancy and are overgrown with large numbers of bacteria.
Bacteria infect insects mostly through the mouth and digestive tract and less commonly through the egg, integument, and tracheae. They may also enter an insect by means of parasites and predators. In the alimentary canal, the bacteria produce enzymes, e.g. lecithinase, proteinase, and chitinase, that damage the midgut epithelium and enable the bacteria to enter the hemocoel.
General Features and Taxonomic Status
- Lack membrane separating the nucleoplasm from the cytoplasm (prokaryotes)
- Peptidoglycan present in the cell walls
- Single celled (unicellular) organism
- Small in size (less than 1 µm to several µm in length)
- Reproduce: asexually by binary fission, sexually by conjugation (transfer of genetic material from one cell to another involving cell-to cell contact)
The bacteria have been placed in the Kingdom Prokaryotae (Buchanan and Gibbons, 1974). Bacteria causing disease in insects are usually grouped as sporeforming pathogens or non-sporeforming pathogens.
Most of the insect pathogenic bacteria occur in the families Bacillaceae, Pseudomonadacea, Entero bacteriaceae, Streptococcaceae, and Micrococcaceae (Tanada and Kaya, 1993).
Family Bacillaceae
Figure
1. H. virescens infected with B. thuringiensis
(Bt).
Larva
killed by Bt.
Midgut
of infected larva (light microscope photograph) showing
cellular degeneration caused by Bt
infection
(healthy midgut tissue for comparison A,
B
and C.
Greatly
enlarged transmission electron micrograph of Bt
sporangium showing a crystal (arrow) and
spore.)
Members of the family Bacillaceae produce endospores; most are gram-positive, motile by lateral or peritrichous flagella (having flagella over the entire surface) or non-motile, and are aerobic, facultative, or anaerobic (Buchanan and Gibbons, 1974). Insect pathogens are found in two genera of this family: Bacillus and Clostridium. Members of the genus Bacillus are aerobic or facultatively anaerobic, while those of the genus Clostridium are anaerobic. Bacillus species usually produce catalase, while species of Clostridium do not produce catalase. The genus Bacillus includes the most promising bacteria for insect control (Tanada and Kaya, 1993). None of the members of the genus Clostridium have been reported as pathogens of heliothines.
In 1915, Berliner in Germany described a sporeforming bacterium, which he named B. thuringiensis; the bacterium had been isolated from a diseased larva of the Mediterranean meal moth. Martin and Travers (1989) obtained more than 1,000 isolates of B. thuringiensis from soils of Africa, Asia, Europe, and North and South America. Bacillus thuringiensis is one of the two bacterial pathogens commercially available. Bacillus popilliae, a causative agent of milky disease of scarabaeid larvae, is the other species of this genus that is commercially available. Bacillus thuringiensis is a gram-positive, soil bacterium characterized by its ability to produce crystalline protein inclusions during sporulation. These proteins are highly toxic to insects and are specific in their activity. In 1956, the first large-scale production (fermentation method) of B. thuringiensis (Thuricide®) was established by Pacific Yeast Products (later to become Bioferm and finally International Minerals and Chemical Corp.).
Bacillus thuringiensis rarely produces epizootics in field populations of insects. For example, the kurstaki strain of B. thuringiensis was isolated from mass-reared H. virescens, but this bacterium does not cause epizootics in this insect. At present, B. thuringiensis is the most widely used bacterium for insect control.
Endotoxin
Most subspecies of Bacillus thuringiensis produce one or more parasporal bodies or crystals in the sporangial cell that are proteinous. It is generally bipyramidal in shape in strains that are mainly active against lepidopterans and may be triangular, cubical, flat, etc. in other subspecies. The parasporal bodies are proteins that require activation by proteolytic enzymes at an alkaline pH to form smaller toxic peptides, the endotoxins. Proteins of bipyramidal crystals range from 120,000 to 230,000 Daltons. A crystal subunit of approximately 230 kDa (kilodaltons) is presently considered to be a dimer (two identical or similar subunits of proteins) of two approximately 130 kDa proteins. In vivo, the dimer (230 kDa) is converted to the lepidopteran protoxin (from 130 to 140 kDa). The protein is proteolytically degraded to a toxin of 64 to 71 kDa. Thus, the main role of the enzymes in the insect's midgut is to degrade the crystal proteins to form the endotoxin (in the midgut pH 10 to 11); the crystal is reduced to a 135 kDa protoxin; the protoxin is reduced by protease to a 66 kDa activated toxin (Tanada and Kaya, 1993). The size of the smallest active fragment, the protease-resistant core, varies with the specific B. thuringiensis protein. The activation process is dependent on the pH and the protease activity and has been shown to account for the spectrum of activity of some B. thuringiensis proteins.
Genetics
of Crystal Proteins
Hofte and Whitely (1989) have proposed a nomenclature and
classification scheme based on the insect specificity
(toxicity) and the primary structure of the proteins. The
Cry I (130-138 kDa) and Cry II (71 kDa) proteins are
primarily toxic to lepidopterans, the Cry III (65-120 kDa)
proteins are known to be toxic to coleopterans, and the Cry
IV (containing toxins of the 128-134 and 72-78 kDa size
classes) proteins are toxic to dipteran insects of the
suborder Nematocera. While most genes/proteins fall into one
of these categories, there are some exceptions (Pietrantonio
et al., 1993).
At present, it is understood that activity of the endotoxin is limited to the insect digestive tract. Susceptible insects have the alkaline pH required for crystal dissolution and the subsequent action of proteolitic enzymes to produce the endotoxin. Reducing substances also play a role in crystal dissolution. The first pathological reaction is paralysis of the gut and mouth parts, leading to cessation of feeding. According to Cooksey (1971), there is a ballooning, exfoliation, and breakdown of the insect's epithelium. Cells of the midgut of intoxicated larvae have enlarged nuclei, alteration of the endoplasmic reticula to vacuole-like configurations, disintegration of the microvilli, and the deformation of the basal infoldings of the epithelium. Along with the intracellular damage, the apical microvilli of midgut cells also swell.
Exotoxins are produced during bacterial growth and are excreted into the medium.
-exotoxin
is a proteinous, thermolabile (decomposed by heat) exotoxin
that is toxic to insects, mice, and other
vertebrates.
ß-exotoxin - (thuringiensin) survives autoclaving (121°C for 15 min) and is highly toxic to larvae of several species of the orders Diptera, Lepidoptera, Coleoptera, Hymenoptera (including bees), Isoptera, Orthoptera, Hemiptera, and Neuroptera. Not all subspecies of B. Thuringiensis produce thuringiensin.
Effects
of the ß-exotoxin on Insects and Other
Invertebrates
Malformation of the mouth parts, antennae, thorax, and wings
is the most apparent sign of the disease. In Drosophila
melanogaster, larval development is retarded and adult
mortality is high, especially in females. Some adults have
atrophied ovaries. The ß-exotoxin affects insects
mainly during metamorphosis. It may kill adult citrus mites
and parasitic plant nematodes.
Effects
of the ß-exotoxin on Vertebrates
ß-exotoxin is toxic to higher animals due to
interference with RNA transcription. Thus the
ß-exotoxin is a toxic metabolite. Because of the
vertebrate toxicity, most commercial preparations of B.
thuringiensis are composed of subspecies that do not
produce thuringiensin.
Along with the discovery of a large number of bacterial subspecies, the number of insects susceptible to B. thuringiensis has also increased. Susceptible insects are mainly in the orders Lepidoptera, Diptera, and Coleoptera. The insects H. zea and H. virescens are susceptible to several subspecies of B. thuringiensis.
Bacillus thuringiensis subspecies affecting lepidopterous larvae appear to be widely distributed. They have been isolated from European, African, Asian, and North and South American soils.
Numerous investigations have shown that lepidopterous insects have the ability to develop resistance to B. thuringiensis.
Bacillus thuringiensis is the most widely used microbial control agent at present and the one most likely to be used even more extensively in the future. Bacillus thuringiensis is being produced by a number of commercial companies for the use against various insect pests associated with public health, forestry, storage products, parks and various recreational areas, and agricultural products. The B. thuringiensis subspecies kurstaki is widely used against lepidopteran insects such as the heliothine species that feed on cotton and tomato.
Non-sporeforming Bacteria -- Serratia marcescens
Figure
2. H. virescens infected with S.
marcescens
S.
marcescens infected
larva.
S.
marcescens infected
adult.
Light
microscope photograph of cross-section of infected larval
midgut showing S. marcescens accumulation above
basement membrane (arrows).
Light
microscope photograph of S. marcescens colonies
originated from infected larvae.
Bucher (1963) classified the non-sporeforming bacterial pathogens of insects into three groups: 1) obligate pathogens, 2) potential pathogens, and 3) facultative pathogens.
Obligate pathogens. These bacteria are very difficult to culture in vitro. In nature, they multiply only within the bodies of host insects where they cause specific diseases. They have a narrow host range. Example: Melissococcus pluton (=Streptococcus pluton) is the causative agent of European foulbrood, which affects honeybees.
Potential pathogens. Such pathogens multiply extracellularly in the hemocoel of insects and produce a lethal septicemia. They grow readily in culture and attack a wide range of insects. Example: Pseudomonas aeruginosa, which infects Heliothis spp. (Bell et al., 1981).
Facultative pathogens. These bacteria possess some mechanism for infecting susceptible body tissues, for damaging host tissue by growing in the gut, and are normally present in the environment. Example: Serratia marcescens has the properties and behavior of a facultative pathogen. There are numerous publications describing ravaging effects of this bacterium on insect colonies including heliothines (Steinhaus, 1959; Bell et al., 1981; Krieg, 1987; Sikorowski and Lawrence, in press).
Both H. zea and H. virescens larvae and adults are often infected with non-sporeforming bacteria mainly in the rearing laboratory where they cause considerable losses, particularly in the younger instars.
The red-pigmented microorganism studied by Bizio (1823) and Sette (1824) that they, respectively, named Serratia marcescens and Zaogalactina imetrofa, were described with what could be considered the original descriptions of S. marcescens.
The enterobacterial genus Serratia is composed of several species, some of which produce prodigiosin, a nondiffusible, water-insoluble red pigment. The majority of strains isolated from man are nonpigmented, and those isolated from insects are red pigmented. However, prodigiosin and prodigiosin-like pigments are produced by many species of bacteria (Vibrio spp., Pseudomonas spp., Alteromonas spp.) and should not be used exclusively as an indicator of Serratia infection.
Serratia species frequently have been recovered from healthy, diseased, or dead insects by numerous workers (Steinhaus, 1959; Bucher, 1963; Bell, 1969; Sri-Arunotai et al., 1975; and Sikorowski, 1985). Serratia marcescens has been isolated from Orthoptera (Schistocerca gregaria; Periplaneta americana), Coleoptera (Melolontha melolontha; Tenebrio molitor), Hymenoptera (Neodiprion lecontii), Lepidoptera (Bombyx mori; H. zea; H. virescens; Malacosoma spp.; Carpocapsa pomonella), and Diptera (Drosophilia sp.; Ceratitis capitata; Dacus dorsalis; Musca domestica) (Krieg, 1987; Thomas and Poinar, 1973). Upon ingestion of high doses of red-pigmented strains, many insect species are susceptible to infection: for example, S. gregaria, N. lecontii, Pristiphora erichsonii, Pieris brassicae, Lymantria dispar, T. ni, Ostrinia nubilalis, and Galleria mellonella are all susceptible. Other pathogenic nonpigmented strains of Serratia have been isolated from S. gregaria, Melanoplus bivittaus, Melolontha spp., Leptinotarsa decemlineata, B. mori, and several noctuids (Krieg, 1987).
Serratia marcescens has been isolated from the inside of eggs of insectary reared H. zea by Bell (1969) and H. virescens by Sikorowski and Lawrence (in press), and from field-collected egg masses of the European corn borer (O. nubilalis) by Lynch et al. (1976). However, S. marcescens is most frequently reported as a pathogen of insectary-reared insects. Thus, S. marcescens is a non-sporeforming facultative pathogen of many insects, including H. zea and H. virescens.
Human safety may also be affected by nonpigmented biotypes of S. marcescens that are encountered with increasing frequency in hospital patients (Neter, 1974), but the broader significance of pigmented biotypes in human infections is still uncertain (Grimont and Grimont, 1978; Tanada and Kaya, 1993). However, organisms considered to be of limited pathogenicity may cause infection in people with grossly reduced vigor, such as prolonged sickness, surgery, etc. (Neter, 1974).
Serratia marcescens infects eggs, larvae, pupae, and adults of heliothines (Sikorowski and Lawrence, in press). Neonate larvae succumb to S. marcescens infection shortly after exposure to the inoculum, within about 24 hours. Older larvae infected with the bacterium usually exhibit a decreasing responsiveness to external stimuli within one to several days after incubation at 29 °C, and death occurs one or two days later. The bacterium multiplies in the hemolymph and causes a lethal septicemia. In our studies, infected larvae became red after death, and only occasionally is a red larva still living. Some larvae infected in the late larval stage pupated and produced red pupae postmortem. Adults may acquire infection by feeding on contaminated food, and they too turn red postmortem. Eventually, the bacterium invades all insect tissues. Serratia marcescens is very infectious and has the capacity to eliminate an entire H. zea or H. virescens colony very quickly, bringing an insect rearing program to a virtual standstill.
Serratia marcescens is also a very important heliothine pathogen in insectaries involved in rearing parasitoids. In bacteria-infected H. zea or H. virescens, the host nutrients are divided among three organisms: heliothine larva, parasitoid larva, and millions of bacterial cells. The host diet also is modified by excretory products of the bacterium. Thus, the number and longevity of parasitoids originating from bacteria-infected heliothines are greatly reduced (Sikorowski et al., 1992).
In general, S. marcescens is not pathogenic to insects when present in the digestive tract in small numbers (Sikorowski, 1985), but once it enters the hemocoel it multiplies rapidly and causes death in one to three days (Sri-Arunotai et al., 1975; Tanada and Kaya, 1993). Sikorowski (1985) reported that he isolated S. marcescens from surface-sterilized, non-feeding pecan weevil larvae (Curculio caryae) following incubation in sterilized soil at 15, 20, 25, or 30 °C for several months (pecan weevils diapause as larvae and do not eat for one to two years). In his opinion, S. marcescens is frequently present in small numbers in many apparently healthy insects and is able to multiply and cause disease if or when larval vigor is greatly reduced.
Neither S. marcescens or Pseudomonas spp. offer much promise as biological control agents, now or for the near future. Some biotypes of S. marcescens have been recognized in clinical materials (Krieg, 1987). The genus Pseudomonas contains a large number of species, most of which are not of medical importance. Of the clinically significant species, P. aeruginosa is by far both the most common and the most important. Other species that may be recovered from patient material include P. fluorescens, P. maltophila, P. putida, P. acidovorans, P. stutzeri, and P. multivorans (synonymous with P. cepacia) (Lindberg, 1974).
Rearing Bacteria-free Heliothines
Eggs from S. marcescens-infected parents may be both externally contaminated and internally infected by this bacterium (Sikorowski and Lawrence, in press). Although to our knowledge, none of the methods used to sanitize eggs eliminate bacteria that are transmitted in the ovum. Surface sanitation of the eggs may provide help in reducing but not eliminating the presence of S. marcescens from an insect colony. For the sterilizing procedure of eggs, see Sikorowski and Goodwin (1985). Pupae originating from larvae infected in the late instars may produce S. marcescens-infected moths. Infected moths may contaminate sugar or honey water (adult diet) and ultimately all moths feeding on it as well as the entire rearing cage (Sikorowski and Lawrence, in press).
Control of bacterial contaminants in various insectaries has been described by many workers (Bell et al., 1981; Sikorowski and Goodwin, 1985; Davis and Guthrie, 1992; and Sikorowski and Lawrence, 1994). Most include in their control measures personal hygiene, maintenance of a clean and sanitary environment, and various methods of sterilizing insectary equipment. Current technology allows us, with less effort, to secure control of bacterial contamination by air filtration, flash sterilization of diet, egg surface sterilization, sterilization and sanitation of equipment, and better trained personnel. In our opinion, rearing bacteria-free insects demands the establishment and enforcement of a strict sanitation program.
S. marcescens and Pseudomonas spp. grow well on many bacteriological media. In our laboratory, S. marcescens was isolated, cultured, and maintained on Serratia medium American Type Culture Collection Medium 1399 (American Ty pe Culture Collection, 1992).
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