Chemical Pesticides: Mode of Action and Toxicology - Chapter 4 ppt

More than 170 genes encoding for Bt toxins have been identified.. • Hemolysins• Enterotoxins • Vegetative insecticidal proteins VIPs • Exoenzymes The genes responsible for the crystal pro

chapter four

Bacillus thuringiensis and

its toxins

This chapter is based mainly on Glare and O’Callaghan (2000), Schnepf et

al (1998), and Crickmore et al (1998) Bacillus thuringiensis (Bt) is now the

most widely used biologically produced pest control agent In 1995

world-wide sales of B thuringiensis were $90 million, representing about 2% of the

total global insecticide market In 1998 nearly 200 insecticidal products were registered in the U.S Studies have so far not shown any pathogenic action

on mammals, birds, amphibians, or reptiles (Board of Agriculture and Nat-ural Resources, 2000)

Bt was first described in 1911 when a bacillus was isolated from the

Mediterranean flour moth, Anagasta kuehniella It is a Gram-positive, rod-shaped, spore-forming bacterium that is closely related to Bacillus cereus,

a bacterium causing gastroenteritis in humans, and to Bacillus anthracis, a

very dangerous bacterium that can be used as a biological warfare agent

The first isolation of Bt was done more than 100 years ago by the Japanese

biologist S Ishiwata, who isolated a bacterium as the causal agent of a disease

of silkworms, but not before 1928, when a project that tried to utilize Bt against a pest, the corn borer (Ostrinia nubilalis), was initiated The presence

of a parasporal body in sporulating Bt cells was noted as early as 1915,

whereas its proteinaceous nature and its toxicity to silkworms were not

recognized before 1954 The potential of Bt as a pesticide was recognized in

the 1920s, and by 1992 more than 40,000 isolates were held in collections throughout the world Today it is more than 60,000 Strains active against a wide variety of insects have now been isolated These include Lepidoptera, Diptera, Coleoptera, Hymenoptera, Hemiptera, and Mallophaga Nematoda and Protozoa are also susceptible to some strains More than 170 genes

encoding for Bt toxins have been identified.

The toxins fall into one of these groups:

• Smaller cytosolic endotoxins (Cyt toxins)

• Hemolysins

• Enterotoxins

• Vegetative insecticidal proteins (VIPs)

• Exoenzymes

The genes responsible for the crystal proteins are called cry genes, and

the crystal toxin is called δ-endotoxin or Cry toxin, whereas the genes

respon-sible for toxic cytosolic proteins (Cyt toxins) are called cyt genes Many Bt

strains are able to produce a number of such smaller cytosolic endotoxins

in addition to the δ-endotoxins, but these are often deposited in inclusion bodies inside the crystals where they can comprise a considerable part of the crystal Unlike the Cry toxins, the other toxins display a broader unspe-cific activity and may have some mammalian toxicity They include the β-exotoxins, hemolysins, and enterotoxins

spectrum of activity, not confined to Diptera It is thermostable and is not destroyed by heating at 70˚C for 15 minutes Because of the vertebrate toxicity, most commercial preparations use subspecies or isolates that do not produce β-exotoxin However, the β-exotoxin can synergize the activity of δ-endotoxin against natural tolerant insects This synergy may result from the inhibitory effect of β-exotoxin on the regeneration of midgut cells dam-aged by δ-endotoxin A new variant of β-exotoxin that is toxic to aquatic mollusks has been described It may be useful to control vectors of schisto-somiasis and other snail-borne diseases, as well as snails in agriculture Hemolysins, which lyse vertebrate erythrocytes, are important virulence factors in vertebrate bacterial pathogens Such toxins are also found in some

Bt strains and seem to be identical to the hemolysin found in B cereus Some

Bt isolates have been found to produce the same type of diarrhea-producing

enterotoxins as B cereus Bt may therefore have implications in causing

gastroenteritis

Bt produces and secretes a number of enzymes, e.g., chitinases, proteases,

and phospholipases, of importance to the pathogenicity They disrupt the per-itrophic membrane, providing access for the true toxins to the gut epithelium

A new class of insecticidal toxins called vegetative insecticidal proteins

has recently been isolated from Bt They are produced during the vegetative

growth stage The proteins are different from other known proteins, and their function and versatility for insect control have yet to be elucidated

4.1 The mechanism of action of δ-endotoxins

conspicuous crystals is the main insecticidal component of B thuringiensis.

At sporulation, the majority of Bt strains produce crystalline inclusions that

contain this insecticidal δ-endotoxin The crystals account for 20% or more

of the total bacterial protein at sporulation and may contain one or several

endotoxins, which differ in activity Many Bt toxin genes and genes for some

endotoxins are encoded by extrachromosomal DNA, often located on large plasmids (A plasmid is a small circular DNA molecule often present in bacterial cells It replicates independently and can be transferred naturally

or artificially to other bacteria.) One plasmid may have genes for more than

one type of endotoxin The cry genes can also be located on the bacterial

chromosome Even genes encoding the same protein may be located both

on plasmids and on the chromosome The amino acid sequences of the toxins are now easier to obtain experimentally than they were earlier; hence, it follows that the classification and nomenclature could be based on sequence similarity that reflects phylogeny, and not the biological activity

The amino acid sequence is used for classification, first in superfamilies Cry1, Cry2, etc If the sequence is more similar, a letter is added (Cry1A, Cry1B, etc.) and the individual proteins are called Cry1A1, Cry3B3, etc Other older classification and designation systems, not based on amino acid sequence, are still in use

Theδ-endotoxins and the other Bt toxins function only as stomach

poi-sons A susceptible insect must eat them, and the crystals dissolve in the midgut This solubilization is sometimes dependent on chymotrypsin-like enzymes The protoxin is further attacked by proteolytic enzymes present

in the gut that convert it to the active toxin An interesting and unexpected finding is that DNA is intimately associated with the toxin crystal and appears to play a role in the proteolytic processing

The active toxin has two main functional entities, responsible for receptor binding and ion channel activity, respectively The activated toxin binds to receptors, which seem to be of different types, on the midgut microvilli of the susceptible insects Different toxins seem to bind to different receptor proteins that may be an enzyme such as aminopeptidase or alkaline phos-phatase, or a cadherin-like membrane protein (The cadherins are proteins that are important in keeping the cells together by mediating Ca+-dependent cell–cell adhesion in animal tissue.) The toxins are anchored to the outer epithelial cell membrane in such a way that the membrane is perforated by pores or channels where ions can freely pass This model proposes that an influx of water, along with ions, results in swelling and lysis The epithelium

is destroyed and the insect rots

Consumption of food treated with the endotoxins, or engineered plants that produce them, results in cessation of feeding of Lepidoptera larvae and paralysis of the gut that retards the passage of food and allows the spores

to germinate The larvae suffer a general paralysis and die Bt

israelen-sis-treated mosquito larvae cease feeding within 1 hour of treatment, show

reduced activity after 2 hours, and general paralysis after 6 hours In beetles death may take longer

Engineering of Cry proteins to create better pesticides is possible A mutant of Cry4B resulted in a threefold increase of toxicity against the mosquito, perhaps by removal of a site sensitive to proteolytic instability More often, increased receptor binding causes the increased efficiency of mutant types

4.2 Biotechnology

Crystal genes have been introduced into other bacteria such as Escherichia

coli, Bacillus subtilis, Bacillus megatorium, and Pseudomonas fluorescens

Fermen-tations of recombinant Pseudomonas have been used to produce concentrated

aqueous biopesticide formulations consisting of Cry inclusions encapsulated

in dead cells Engineered forms of Cry proteins may show improved potency

or yield and may make them a more attractive and practical alternative or supplement to other traditional pesticides The gene has also been

intro-duced into a bacterium (Clavibacterium xyli) that lives inside plants When

corn is infested with this bacterium, the crop is protected against corn borer

Other endophytic microorganisms (Azospirillum spp., Rhizobium, etc.) have also been engineered, and cry genes specifically active to Diptera have been

introduced into other bacteria, including Cyanobacteria

4.3 Engineered plants

In order to get an optimal production of toxins in plants, the cry gene from

Bt must be modified extensively before being introduced into the plant’s

genome, and only the part of the gene that codes for the active part of the

Bt toxin is used Full-length unmodified cry genes give quite inefficient toxin

production Varieties of potato, cotton, corn, and many other plants

contain-ing modified cry genes are now available on the market in some countries.

The marketing started in 1996 and the proponents for such plants are very enthusiastic Because the toxins are produced continuously and apparently persist for some time in the plant tissue, fewer, if any at all, chemical pesti-cides must be used Beneficial insects are not harmed However, the public opinion, notably that of environmentalists, is skeptical There are fundamen-tal beliefs that such gene manipulations are to play the role of God, or are

to intervene with natural selection in an unethical and unacceptable way Other more trivial arguments may be more relevant to the rational mind, i.e., that there may be some unknown health or ecological effects It is outside my field

of knowledge to offer a qualified opinion about these important questions

4.4 Biology

A biologist will want to know something about the relationship of Bt to other

bacteria, the natural function, if any, of the toxins, as well as the natural

occurrence of the Bt strains Numerous strains have been isolated from a

wide range of habitats such as soil, mushroom compost, and stored products

Bt may be present in samples from beach deserts and tundra, and may

multiply in cadavers of insects and other animals Insecticidal activity does not correlate with the origin of the isolate, and many of them do not seem

to be toxic for insects Bt may be regarded as a ubiquitous soil microbe and

common on vegetation Their spores may be viable for years in the soil

environment Of viable bacterial spores present on foliage on various

decid-uous and conifer trees, Bt often accounts for 30 to 100% Viable spores are

also quite common on grasses and herbs The natural function of the toxins may be connected to this habitat If insects visiting the plant surfaces are infected, they are killed by the toxins and make an excellent food source for the bacterium

As mentioned, Bt makes a taxonomic group together with Bacillus

anthra-cis and B cereus Bt and B cereus are so closely related, both serologically

and by various methods using DNA sequences, that they may be regarded

as one species The much feared toxin of B anthracis is different from the δ-endotoxin of Bt The B anthracis toxin produces three factors — lethal

factor, protective antigen, and edema factor — that are encoded by three different genes The protective antigen recognizes and binds to certain recep-tors on the cell membrane, where they form a pore The lethal factor and edema factor bind to the protective antigen and are taken into the cell by a process called endocytosis After uptake, the lethal factor acts as a protease that specifically splits an enzyme important for internal signaling in the cell (MAP kinase, or MAPKK1 and MAPKK2) MAP kinase is an abbreviation

of “mitogen-activated protein kinase,” which is described in Alberts et al (2002) and other cell biology textbooks The edema factor acts also as an enzyme (adenylate cyclase) that inhibits immune response The anthrax toxins are therefore different from the δ-endotoxin of Bt, although the making

of pores is a related mechanism

4.5 Commercial products

The first commercial product that appeared in 1938 for use against lepi-dopterous larvae was Sporeine, which was produced by several companies Names of other old products are Dipel WP, a wettable powder, and Dipel

LC, a liquid concentrate used for aerial low-volume application The

Bt-based microbial pesticides have so far been the most successful

biopesti-cides The rapid growth of Bt-based pesticides is occurring as replacements

of chemicals that were banned or phased out in environmentally sensitive areas, in consumer and export markets in which concerns about food residue are high, and in organic food production The current sales are at least $140 million Today the commercial products are based on various strains of the more than 26 different subspecies

The several strains of the subspecies Bt kurstaki, or strains of Bt kurstaki with various cry genes from Bt aizowai, Bt morrisoni, and Bt kumatoensis, are

marketed for use against lepidopterous larvae, or in some cases against

Colorado beetles Bt is now sold under about 30 trade names Preparations with Bt tenebrionis are used against Colorado beetle, and Bt japonensis may be

used against soil-inhabiting beetles in turf and ornamentals Even more

inter-esting is Bt israelensis, which is used in aerial applications against mosquito and

blackfly larvae It is also sold under many different trade names

Various preparations of δ-endotoxin (Cry1A(c), Cry1C, or Cry3A) are

also marketed The toxins are produced by engineered Pseudomonas

fluo-rescence and are formulated as microcapsules or as granular formulations.

They are used against Lepidoptera, armyworms, Colorado beetles, and corn borers

National approval authorities in some countries hesitate to approve Bt preparations because of the similarities between B thuringiensis and the pathogenic species B anthracis and B cereus However, no ecotoxicological

or human toxicity seems to be associated with Bt itself or its toxins The

half-life in soil is short, and it is destroyed by sunlight The questions about

possible ecological consequences of Bt plants are not yet settled.