Classification of Cry toxins according to their insect host specificities proposed by Crickmore et al. .
The toxins of Bacillus thuringiensis (Bt) have shown great potential in the control of harmful insects affecting human health and agriculture, used as the main biological agent for the formulation of bioinsecticides due to its specificity to target different insects’ orders. This has led Bt-based products to become the best-selling biological insecticides in the world since the genes encoding insecticidal proteins have been successfully used in novel insecticidal formulation, genetically engineered (GE) crops, and development of transgenic rice that produce insecticidal toxins derived from Bacillus thuringiensis. It has been proven that insecticidal activity of Bt protein crystals can prolong their toxicity in shelf life or field under specific conditions, and this can improve the use of special strains and formulations to control insect vectors of diseases. Bt toxins have shown well-documented toxicity against lepidopterans, coleopterans, hemipterans, dipterans, nematodes, Rhabditida and human cancer cells of various origins. These crystal toxins may be responsible for other novel biological properties suggesting a pluripotential nature with different specificities.
- Bacillus thuringiensis
- Cry toxins
In the modern era,
Bt toxins have been applied to the environment since 1933 and began to be used commercially in France in 1938, and by 1958 their use had spread to the United States. From the 1980s Bt becomes a pesticide of global interest .
Bt crystal and secreted soluble toxins are highly specific for their hosts and have gained worldwide importance as an alternative to chemical insecticides. Bt toxins have been considered as the most successful bioinsecticide during the last century. Currently, it consists of more than 98 (424 million USD) of formulated sprayable bacterial pesticides  and is the most common environmental-friendly insecticide used and is the basis of over 90% of the pesticides available in the market today .
2. Bioinsecticide activity of
Bacillus thuringiensis proteins
The main difference between
Bt strains synthesize crystal (Cry) and cytolytic (Cyt) toxins (also known as δ-endotoxins), at the onset of sporulation and during the stationary growth phase as parasporal crystalline inclusions. Additionally, Bt isolates can also synthesize other insecticidal proteins during the vegetative growth phase; these are subsequently secreted into the culture medium, the vegetative insecticidal proteins (Vip) [5, 16], and the secreted insecticidal proteins (Sip) .
|Main classes||Order||Cry toxins|
|Group 1||Lepidoptera||Cry1, Cry9, and Cry15|
|Group 2||Lepidopteran and dipterous||Cry2|
|Group 3||Coleoptera||Cry3, Cry7, and Cry8|
|Group 4||Diptera||Cry4, Cry10, Cry11, Cry16, Cry17, Cry19, and Cry20|
|Group 5||Lepidoptera and Coleoptera||Cry1I|
However, this nomenclature was not ideal, since the new toxins had to be tested against an increasing number of insects so that the toxin and the gene could be named; that was when the
Cry proteins have been reported to be toxic to Lepidoptera, Coleoptera, Hymenoptera, Hemiptera, Diptera, Orthoptera, and Mallophaga and also against nematodes, mites, and Protozoa (Figure 1) . Some toxins have an expanded spectrum of action to two or more order or phylum . For example, Cry1B is one of those that present a remarkable activity against larvae of Lepidoptera, Diptera, and Coleoptera. So, the combination of toxins present in a strain will define its spectrum of action .
In contrast, Cyt toxins have predominant activity against dipterous; however, they have toxic activity against some lepidopteran and coleopteran ; in addition, some Cyt toxins are able to establish synergy for insecticidal activity with other Bt proteins such as Cry or Vip3 and to reduce the resistance levels of Cry proteins toward some insect species of the Coleoptera and Diptera orders (Figure 1). The Cyt1Aa toxin from
2.1 Bti toxins
Bti serovariety, H-14, is a subspecies of the diversified
Bti toxin Cry4Ba is active primarily against
All Bti insecticidal proteins are produced as protoxins, and all must be activated in vivo by insect midgut proteases prior insecticidal activity.
2.2 Mechanism of Cry toxin action
Although the mechanism of action of Cry toxins against various insects has been widely investigated, there are still many controversies. Therefore, there are currently different models in the literature that seek to explain it .
The sequential union model is known as the classical mechanism. It has been detailed in studies with the Cry1Ab protein in
The second proposed mechanism called signaling pathway model has similarities with the previous model; however, in this other causes for cell death are assigned. According to this theory, Cry proteins affect the cell in two ways: first by the formation of pores in the membrane, as mentioned in the sequential binding model and, second, by the production of successive reactions that alter the cellular metabolism. According to this hypothesis, Cry toxins bind to cadherin receptors, which stimulate heterotrimeric G protein and adenylyl cyclase with an increase in cAMP production. The cAMP activates the protein kinase A, which stimulates apoptosis with an activation of the Mg2+ channels in the plasma membrane. The opening of these channels causes an abnormal movement of the ions in the cytosol, stimulating the process of apoptosis (Figure 3) [1, 3, 30].
The germination of the spores also contributes to the death of insect, since the vegetative cells can replicate within the host’s hemolymph and cause septicemia; however, the δ-endotoxins alone are sufficient to kill some insect species if they are produced in high doses. This feature has been exploited by expressing the delta endotoxin genes in bacteria that better adapt to a particular environment, as well as its expression in genetically modified plants [31, 32].
3. Howard T. Dulmage’s methods and contributions on
Howard T. Dulmage was a microbiologist who established his line research in the study of pathogenic bacteria insects  and is considered one of the most important pioneers in the development of technologies for the implementation of
Working at the US Department of Agriculture, at the Agricultural Research Service (USDA-ARS), in Brownsville, Texas, Howard T. Dulmage from the pink worm,
Strain HD-1 is one of the best-studied strains, since it is characterized by the carrying of a variety of Cry anti-Lepidoptera genes,
H. Dulmage sets up the basis for the fermentation and formulation procedures of Bt culture extracts for their commercialization  and were among the most important pioneers in the development of technologies for the implementation of Bt as a biological pest control agent. He established diverse methodologies for mass production product formulation and power standardization .
At the beginning of the 1970s, two great advances were obtained by Dulmage, the first was based on the recovery of the spore-crystal complex by means of precipitation with lactose-acetone to produce powders and wettables, which was rapidly developed and adapted in the industry. The second was the adoption of a standardized system to calibrate the potency of the different preparations of
In 1984, Dulmage participated in the establishment of a bioassay protocol for toxicity assessment of
Specifies a standard cup for larval exposure to Bti extracts.
Establishes a number of 20 larvae per cup and three replications for the concentrations assayed.
If a minimum of six extract concentrations is tested, a repetition of the assay is required.
A computational probit analysis is required for evaluating the toxicity as LC50.
A mortality or pupation higher than 5% in the control invalidates the bioassay.
Additionally, the study suggested a variability coefficient of less than 20% for each repetition. Dulmage, together with a team of colleagues, tested the validity of this protocol and suggested some considerations for the management of the reference standard strains and for the establishment of new ones.
3.1 Howard T. Dulmage’s fermentation extracts
From 1970 to 1988, Dulmage established the largest Bt collection in the Americas, and he collected more than 800 isolates that were named using his HD code, belonging to 21 serovarieties. From these 800 isolates, 17 belonged to the H-14 serovariety, corresponding to Bti. He conducted a series of fermentation experiments with Bt in order to optimize the production and to assess the effectiveness of powder; hundreds of fermentation extracts were generated, and some of them were donated by the US Department of Agriculture in 1989 to the International Collection of Entomopathogenic Bacillus of the Faculty of Biological Sciences of the University of Nuevo León, Mexico, which has approximately 4000 stored fermentation extracts of which 3000 of them correspond to HD strains, and currently extracts are found in the form of dry powder, with different times of storage .
3.2 Bti strain collection
In the 1970s, Dulmage continued to the control of disease-transmitting mosquito larvae using lepidopteran-active isolates having some reported dipteran activity. When Dulmage became aware of the discovery of a new Bt subspecies capable of attacking dipteran larvae, especially simuliids (
One of the greatest contributions of Dulmage to Bti research was the compilation of a protocol guide for Bt H-14 serovariety local production. This guide was an extension of the procedures developed by him for the production, formulation, and standardization of lepidopteran-specific serovarieties. These guidelines were presented and discussed in the informal consultation on local H-14 Bt production, in Geneva, Switzerland, in October 1982. The 128-page booklet was prepared by Dr. Dulmage, at the request of the Scientific Working Group on biological control of vectors of the Special Program for Research and Training in Tropical Diseases of the World Health Organization, and was published in 1983 .
In 1985, Dulmage and a research group proved the tested strain was Bti HD-968-S-1983, which resulted to be 4.74 times more potent than the standard use (IPS-78); the potency assigned to it was 4740 ± 398 ITU/mg. They recommended the use of this strain as the potency reference standard for comparison with any Bti formulation.
Twenty samples of the strain HD-500 and HD-567 of Bti fermentation extracts from the collection of Dulmage et al.  recovered by lactose-acetone coprecipitation during the period from 1978 to 1983 maintained their residual toxic activity against the mosquito
Bti protein crystals from fermentation extracts showed persistence of toxic activity of fermentation extracts after more than three decades. This opens the possibility of improving the use of special strains and improved formulations to control insect vectors of diseases.
4. New Cry toxins
Despite the success of the application of Bt crystal proteins for the biological control of pests, at present it is still necessary to identify new Cry toxins with greater toxicity; this approach is considered one of the best ways to counter the potential resistance evolved by insects as well as in developing products against a wider spectrum of insect pests. Traditionally, Bt isolates were screened for their insecticidal spectrum by the time-consuming and laborious insect bioassays [22, 46]. Since only a limited number of cry genes have been used for insect control either in sprays or transgenic crops so far, novel insecticidal genes are required .
The most common technique used to predict toxicity is the polymerase chain reaction (PCR), through the identification of new cry genes , but high-throughput sequencing technology has also been used in the discovery of toxins . Seventy-two antigenic groups (serovariety) have been distinguished for
Additionally, the construction of
Moreover, a combination of genomics, transcriptomics, proteomics, and metabolomics could be used to study
Furthermore, recent studies have confirmed more new potentials of different Bt strains. These new features are including plant growth promotion , bioremediation of heavy metals and other chemicals [1, 52], anticancer activities , polymer production , and antagonistic effects against plant and animal pathogenic microorganisms .
Bacillus thuringiensis development on rice crops
Genetically engineered or transgenic crops producing Cry proteins from
Bt crops produce either a single toxin or more than one Bt toxin; these are called pyramided crops. Bt pyramided crops delay evolution of resistance to target pests, insects resistant to one toxin are killed by other toxins in the pyramid [57, 58]. Nevertheless, pyramided Bt crops are vulnerable to the development of cross-resistance. The use of Bt pyramids and the simultaneous planting of non-Bt crops are the main strategies applied to produce susceptible pest insects (known as the “refuge strategy”) .
Rice is a primary food source for more than half of the world’s population making it one of the most fundamental crops. Since 1989 multiple insect-resistant genetically engineered (IRGE) rice lines expressing
Since Cry1Ab was first introduced into a japonica rice variety, many Bt genes have been found, and only a few of them were selected for developing transgenic crops . Because deploying two or more Bt genes in one rice variety can delay the emergence of pest resistance [64, 74], scientists started to develop Bt hybrid rice lines with Cry1Ab/Cry1Ac into various rice plants which have both high grain yield and good grain quality .
Some advantages of expressing fusion proteins like Cry1Ab/Cry1Ac and Cry1Ab/Vip3A are the equalization of the expression level of the two proteins, trait integration in different crops, and highly efficient expression strains . Studies on Cry1Ab/Cry1Ac fusion protein have demonstrated great effectiveness significantly reducing the incidence of
The rice water weevil (
Some of the strategies to control this insect pest are the use of pyrethroids, which are toxic to aquatic organisms , synthetic insecticides, and weed control around fields to reduce habitat for rice water weevil adults.
6. Resistance to
The use of transgenic plants has greatly increased the selection pressure on target pest populations and is likely to become much more acute in natural conditions if
In agriculture worldwide, repeated applications of Bt sprays and widespread adoption of Bt crops (transgenic crops protected from insects by the expression of
Field populations of
Resistance to Cry toxins can be developed by mutations in the insect pests that affect any of the steps of the mode of action of Cry toxins . “Field populations” refers to insects on the field, since the conditions are distinct in vitro, can be developed by different mechanisms, such as altered activation of Cry toxins by midgut proteases sequestering the toxin by glycolipid moieties or esterases, by inducing an elevated immune response, and by alteration resulting in reduced binding to insect gut membrane; among all these mechanisms of resistance, the most common mechanism of toxin resistance is the reduction in toxin binding to midgut cells, which in different resistant insect species include mutations in Cry toxin receptors such as cadherin (CAD)-like proteins, alkaline phosphatase (ALP), or aminopeptidase N (APN) or mutations in the ABCC2 transporter .
The emergence of resistant insects is a problem that both
There are different methods to counteract the resistance of insects to Bt toxins, for example, assisted mutation with UV light; the combination of Bt toxins with other toxins, such as
Nevertheless, a new method has been used to combat resistance to Bt toxins, the phage-assisted continuous evolution (PACE), which rapidly evolves Bt toxins to bind a new receptor with high affinity and specificity, expressed on the surface of insect midgut cells. The PACE system enhances the insecticidal activity against both sensitive and Bt-resistant insect larvae up to 335-fold, through more than 500 generations of mutation, selection, and replication to bind a new receptor . Collectively, these methods establish an approach to overcoming Bt toxin resistance.
7. Formulations based on
The production of toxic proteins has given
The wide variety of formulations based on spores and crystals intended for being ingested by the white insect are the result of many years of research. The development of a large variety of spore-crystal complex matrices allows for improvements, such as increased toxic activity, increased palatability to insects, or longer storage times. These matrices use chemical, vegetable, or animal products, which are constituted in such a way that they favor contact between crystals and insects, without harming humans or the environment .
Proper formulation can help to overcome several of the factors that limit or reduce its larvicidal activity and improve control performance by enabling greater contact with target larvae, ensuring stability under storage and field conditions, providing a variety of application options, and increasing the ease of handling. There are several types of formulations, among the most used are:
Formulated by sorption of an active ingredient on finely ground mineral powder (talc, clay, etc.).
Particle size of 50–100 μm.
Powders can be applied directly to the target, either mechanically or manually.
The inert ingredients for this formulation are anticaking agents, ultraviolet protectors, and adhesive materials to improve adsorption.
Concentration of the active ingredient (organism) in the powder is usually 10%.
Granular particles are larger and heavier than powder formulations.
Particle size coarse of 100–1000 μm for granules and 100–600 μm for microgranules.
Made of mineral materials (kaolin, attapulgite, silica, starch, polymers, dry fertilizers, and residues of ground plants) .
Concentration of the active ingredient (organisms) in granules ranges from 5 to 20%.
Once applied, the granules slowly release their active ingredient.
Finely ground dry formulations that will be applied after suspension in water.
Produced by mixing an active ingredient with surfactants, wetting and dispersing agents, and inert fillers, followed by milling.
Particle size approximately 5 μm.
Long storage stability, good miscibility with water, and convenient application with conventional spray equipment .
Designed to be suspended in water.
The granules break to form a uniform suspension similar to that formed by a wettable powder.
Compared to powdered products, these WGs are relatively dust-free and with good storage stability.
The products contain a wetting agent and dispersing agent similar to those used in wettable powders, but the dispersing agent is usually at a higher concentration.
Consist of liquid droplets dispersed in another immiscible liquid.
Size of the droplets in the dispersed phase varies from 0.1 to 10 μm.
The emulsion can be oil in water (EW), which is a normal emulsion, or water in oil (EO), an inverted emulsion. Both products are designed to be mixed with water before use.
A mixture of a finely ground solid active ingredient dispersed in a liquid phase, usually water.
The solid particles do not dissolve in the liquid phase, so that the mixture needs to be stirred before application to keep the particles evenly distributed.
The composition of the suspension concentrate is complex and contains wetting/dispersing agents, thickening agents, antifoaming agents, etc., to ensure the required stability.
They are produced by a wet milling process.
Particle size distribution of 1–10 μm.
Dispersions of solid active ingredients in a nonaqueous liquid intended for dilution before use.
The nonaqueous liquid is more often an oil (vegetable oil).
Oil dispersion provides several important characteristics, such as the ability to supply water-sensitive active ingredients and the ability to use an adjuvant fluid instead of water that can increase and extend pest control.
Stable suspension of microencapsulated active ingredient in an aqueous continuous phase.
Intended for dilution with water before use.
The bioagent as an active ingredient is encapsulated in capsules (coating) made of gelatin, starch, cellulose, and other polymers.
Protected from extreme environmental conditions (UV radiation, rain, temperature, etc.).
Residual stability increases due to slow (controlled) release.
The most frequently applied encapsulation method uses the principle of interfacial polymerization.
The extension of pesticide formulations containing Bt will depend essentially on our capacity to improve the performance of the products used . Therefore, biotechnology companies have the task of providing not only formulations adapted to certain crops and insect pests, but also, they must look for and produce bioinsecticides based on the new high-potency strains originating from the agroecosystems where they are going to apply. It is expected that the new products that appear in the market will provide a spectrum of higher activity that will impact on a greater number of pests in other crops and can help develop sustainable agriculture .
8. Bioinsecticides based on
Worldwide, the use of biopesticides increases 16% annually, which represents approximately 8% of the pesticide trade in the world . The formulations derived from natural materials such as bacteria, animals, plants, or minerals offer a powerful tool to create a new generation of sustainable products . About 90% of microbial biopesticides are derived from a single entomopathogenic species
The varieties of
|Susceptible insects||δ-Endotoxin||Producer company|
|Lepidoptera||Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab||Abbott-Dupont and Certis|
|Lepidoptera||Cry1Aa, Cry1Ab, Cry1Ba, Cry1Ca, and Cry1Da||Abbott-Dupont and Kenogard|
|Coleoptera||Cry3Aa||Thermo Trilogy, Columbia MD, Certis Mycogen, and Novo Nordisk|
|Diptera||Cry4A, Cry4B, Cry11A, and Cyt1Aa||Abbott-Dupont, Novo Nordisk, and Certis|
More than a century after its discovery,
These new environmental features include the toxicity against nematodes, mites, and ticks, antagonistic effects against plant and animal pathogenic bacteria and fungi, plant growth-promoting rhizobacteria (PGPR) activities, bioremediation of different heavy metals and other pollutants, biosynthesis of metal nanoparticles, production of polyhydroxyalkanoate biopolymer, and anticancer activities (due to parasporins) [51, 52, 53].
Toxicity against nematodes with several classes of Cry toxin (Cry5, Cry6, Cry13, Cry14, Cry21, and Cry55) is well established. In addition to these Cry proteins, thuringiensin, chitinase, and a metalloproteinase from
Cry proteins synthesized by
Some strains of
Different strains of
Parasporins are a heterogenous group of Cry proteins produced by noninsecticidal
10. Advantages and disadvantages
The biopesticide based on bacteria is probably the most used and is cheaper than the other methods of bioregulation of pests . Almost 90% of the microbial biopesticides that are commercially available are
|Application with difficulty|
|It is not easy to produce it|
|Little diffusion and acceptance by producers|
|Its quality could not be controlled. Sometimes it works, and sometimes it does not|
|Variability in insect resistance|
|Location. Its use may be limited to faunas of a certain region|
During the last two decades, new methods have been widely used on Bt to overcome resistance to insects, and it is expected that this advancing trend will be well continued in the future, including the search for new toxins and strains with increased toxic activity and the development of new biopesticides and technologies to maintain the success of this bioinsecticide which is a great challenge to overcome.
Nowadays there exist different lines of research that seek to use
Conflict of interest
The authors declare that they have no conflicts of interest.
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