**2. Bioinsecticide activity of** *Bacillus thuringiensis* **proteins**

The main difference between *Bacillus thuringiensis* and other closely related bacillus is the formation, during the sporulation process, of one or more crystalline bodies of a protein nature adjacent to the spore. Some of these parasporal crystals known as δ-endotoxins (Cry and Cyt) confer the pathogenic capacity against larvae of different orders of insects, mostly Lepidoptera, Diptera, Coleoptera and in some cases against species of other phyla [14]. By synthesizing parasporal crystalline inclusion during sporulation, the bacterium can ensure its survival, since a dead insect can provide sufficient nutrients that allow the spores to germinate [15].

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) [17].

This part refers to the nomenclature first used for Cry genes, on the next part of the page it explains the nomenclature currently used for Bacillus thuringiensis genes [18] (**Table 1**).


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

### **Table 1.**

*Classification of Cry toxins according to their insect host specificities proposed by Crickmore et al. [18].*

**185**

**Figure 1.**

*Hemiptera, and Hymenoptera [15, 21, 23].*

*Toxic Potential of Bacillus thuringiensis: An Overview DOI: http://dx.doi.org/10.5772/intechopen.85756*

they share more than 95% pairwise identity [19].

genes [21].

named; that was when the *Bacillus thuringiensis* Toxin Nomenclature Committee was created in 1993 and proposed a new classification system [18], which consists of giving the new toxin a four-rank name depending on its degree of pairwise amino acid identity to previously named toxins, using Arabic numbers for the first and fourth rank and uppercase and lowercase letters for the second and third ranks, respectively, for example, Vip1 and Vip2 if they share less than 45% pairwise identity, Vip3A and Vip3C if they share less than 78% pairwise identity, Vip3Aa and Vip3Ab if they share less than 95% pairwise identity, and Vip3Aa1 and Vip3Aa2 if

Based on the amino acid sequences, there are 75 families of Cry proteins, with 800 different *Cry* genes [20], while the Cyt proteins consist of three families with 38

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**) [22]. Some toxins have an expanded

*Insecticidal activity of Cry and Cyt δ-endotoxins against the orders Diptera, Coleoptera, Lepidoptera,* 

*Toxic Potential of Bacillus thuringiensis: An Overview DOI: http://dx.doi.org/10.5772/intechopen.85756*

*Protecting Rice Grains in the Post-Genomic Era*

cancer cells [2, 6–10].

the market today [13].

to germinate [15].

genes [18] (**Table 1**).

**Main classes**

δ-endotoxin. Crystalline inclusions from Bt are showing well-documented toxicity to a wide variety of insect pests, such as Lepidoptera, Coleoptera, and Diptera [5], hemipterans, as other biological activities such as molluscicidal, nematicide (human and animal parasites, and free living; Rhabditida), acaricide and even against human

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

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 [12] and is the most common environmentalfriendly insecticide used and is the basis of over 90% of the pesticides available in

The main difference between *Bacillus thuringiensis* and other closely related bacillus is the formation, during the sporulation process, of one or more crystalline bodies of a protein nature adjacent to the spore. Some of these parasporal crystals known as δ-endotoxins (Cry and Cyt) confer the pathogenic capacity against larvae of different orders of insects, mostly Lepidoptera, Diptera, Coleoptera and in some cases against species of other phyla [14]. By synthesizing parasporal crystalline inclusion during sporulation, the bacterium can ensure its survival, since a dead insect can provide sufficient nutrients that allow the spores

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)

This part refers to the nomenclature first used for Cry genes, on the next part of the page it explains the nomenclature currently used for Bacillus thuringiensis

However, this nomenclature was not ideal, since the new toxins had to be tested

Group 4 Diptera Cry4, Cry10, Cry11, Cry16, Cry17, Cry19, and Cry20

*Classification of Cry toxins according to their insect host specificities proposed by Crickmore et al. [18].*

against an increasing number of insects so that the toxin and the gene could be

**Order Cry toxins**

Group 1 Lepidoptera Cry1, Cry9, and Cry15

Group 3 Coleoptera Cry3, Cry7, and Cry8

Group 2 Lepidopteran and dipterous Cry2

Group 5 Lepidoptera and Coleoptera Cry1I Group 6 Nematodes Cry6

States. From the 1980s Bt becomes a pesticide of global interest [11].

**2. Bioinsecticide activity of** *Bacillus thuringiensis* **proteins**

[5, 16], and the secreted insecticidal proteins (Sip) [17].

**184**

**Table 1.**

named; that was when the *Bacillus thuringiensis* Toxin Nomenclature Committee was created in 1993 and proposed a new classification system [18], which consists of giving the new toxin a four-rank name depending on its degree of pairwise amino acid identity to previously named toxins, using Arabic numbers for the first and fourth rank and uppercase and lowercase letters for the second and third ranks, respectively, for example, Vip1 and Vip2 if they share less than 45% pairwise identity, Vip3A and Vip3C if they share less than 78% pairwise identity, Vip3Aa and Vip3Ab if they share less than 95% pairwise identity, and Vip3Aa1 and Vip3Aa2 if they share more than 95% pairwise identity [19].

Based on the amino acid sequences, there are 75 families of Cry proteins, with 800 different *Cry* genes [20], while the Cyt proteins consist of three families with 38 genes [21].

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**) [22]. Some toxins have an expanded


### **Figure 1.**

*Insecticidal activity of Cry and Cyt δ-endotoxins against the orders Diptera, Coleoptera, Lepidoptera, Hemiptera, and Hymenoptera [15, 21, 23].*

spectrum of action to two or more order or phylum [10]. 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 [4].

In contrast, Cyt toxins have predominant activity against dipterous; however, they have toxic activity against some lepidopteran and coleopteran [24]; 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 *Bacillus thuringiensis* var. israelensis is active against *Chrysomela scripta* and *Culex quinquefasciatus* and can prevent the development of resistance to the proteins Cry3Aa, Cry4, and Cry11Aa [14].

### **2.1 Bti toxins**

*Bacillus thuringiensis subsp. israelensis* (Bti) was first isolated from a water pond in the Negev desert [25] and was the very first strain described for having insecticidal activity outside Lepidoptera.

Bti serovariety, H-14, is a subspecies of the diversified *Bacillus thuringiensis* species. The serovariety H-14, Bti, produces four main toxins (Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa) specific to dipterans (mosquitoes, blackflies, etc.) which represent a serious threat to public health because of their hematophagous nature and vector capacity responsible for high morbidity and mortality in billions of people spread over almost half of the planet.

Bti toxin Cry4Ba is active primarily against *Anopheles* and *Aedes* and shows no toxicity to *Culex* species, in contrast to Cry4Aa toxin that is toxic to *Culex* larvae. Cry11 is the most toxic to Aedes, and Cyt1Aa shows low (*Aedes*, *Culex*) to non-toxicity at all (*Anopheles*). Cyt1Aa has a strong synergistic effect on the toxicity of Cry toxins in all mosquitoes. In addition to its own mosquitocidal and cytotoxic activity, Cyt1A was shown to act synergistically with the other Bti toxins [26, 27].

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 [28].

The sequential union model is known as the classical mechanism. It has been detailed in studies with the Cry1Ab protein in *Manduca sexta*. It postulates that the toxic properties come from crystalline inclusions produced during the sporulation of *Bt*. The crystals and their subunits are inert protoxins and are not biologically active, and their mode of action can be plotted as follows: the δ-endotoxins are ingested, the crystals are solubilized by the alkaline pH of the intestine, the inactive protoxins are digested by proteases of the midgut which produces an active toxin of about 60–70 kDa resistant to proteases, and then the Cry toxins come into contact with the N-aminopeptidase receptors and cadherin on the surface of the membrane. The affinity between toxins and certain types of receptors results in proteolysis of the Cry protein that causes structural changes in the chains and forms oligomers that function as "pre-pores." The N-aminopeptidase receptor anchors the pre-pore in the lipid bilayer, pore formation affects integrity of the membrane,

**187**

(**Figure 2**) [1, 29].

**Figure 3.**

**Figure 2.**

*Toxic Potential of Bacillus thuringiensis: An Overview DOI: http://dx.doi.org/10.5772/intechopen.85756*

*Mechanism of action of Cry proteins according to the sequential binding model.*

and electrophysiological evidence and biochemistry suggest that the pores cause an osmotic imbalance that causes cell death and lysis; the intestine is paralyzed, the

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

insect stops feeding, and there is diarrhea, total paralysis, and finally death

*Mechanism of action of Cry proteins according to the signaling pathway model.*

*Toxic Potential of Bacillus thuringiensis: An Overview DOI: http://dx.doi.org/10.5772/intechopen.85756*

### **Figure 2.**

*Protecting Rice Grains in the Post-Genomic Era*

the proteins Cry3Aa, Cry4, and Cry11Aa [14].

people spread over almost half of the planet.

**2.2 Mechanism of Cry toxin action**

in vivo by insect midgut proteases prior insecticidal activity.

rently different models in the literature that seek to explain it [28].

cidal activity outside Lepidoptera.

spectrum of action [4].

**2.1 Bti toxins**

toxins [26, 27].

spectrum of action to two or more order or phylum [10]. 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

In contrast, Cyt toxins have predominant activity against dipterous; however, they have toxic activity against some lepidopteran and coleopteran [24]; 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 *Bacillus thuringiensis* var. israelensis is active against *Chrysomela scripta* and *Culex quinquefasciatus* and can prevent the development of resistance to

*Bacillus thuringiensis subsp. israelensis* (Bti) was first isolated from a water pond in the Negev desert [25] and was the very first strain described for having insecti-

Bti serovariety, H-14, is a subspecies of the diversified *Bacillus thuringiensis* species. The serovariety H-14, Bti, produces four main toxins (Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa) specific to dipterans (mosquitoes, blackflies, etc.) which represent a serious threat to public health because of their hematophagous nature and vector capacity responsible for high morbidity and mortality in billions of

Bti toxin Cry4Ba is active primarily against *Anopheles* and *Aedes* and shows no toxicity to *Culex* species, in contrast to Cry4Aa toxin that is toxic to *Culex* larvae. Cry11 is the most toxic to Aedes, and Cyt1Aa shows low (*Aedes*, *Culex*) to non-toxicity at all (*Anopheles*). Cyt1Aa has a strong synergistic effect on the toxicity of Cry toxins in all mosquitoes. In addition to its own mosquitocidal and cytotoxic activity, Cyt1A was shown to act synergistically with the other Bti

All Bti insecticidal proteins are produced as protoxins, and all must be activated

Although the mechanism of action of Cry toxins against various insects has been

widely investigated, there are still many controversies. Therefore, there are cur-

The sequential union model is known as the classical mechanism. It has been detailed in studies with the Cry1Ab protein in *Manduca sexta*. It postulates that the toxic properties come from crystalline inclusions produced during the sporulation of *Bt*. The crystals and their subunits are inert protoxins and are not biologically active, and their mode of action can be plotted as follows: the δ-endotoxins are ingested, the crystals are solubilized by the alkaline pH of the intestine, the inactive protoxins are digested by proteases of the midgut which produces an active toxin of about 60–70 kDa resistant to proteases, and then the Cry toxins come into contact with the N-aminopeptidase receptors and cadherin on the surface of the membrane. The affinity between toxins and certain types of receptors results in proteolysis of the Cry protein that causes structural changes in the chains and forms oligomers that function as "pre-pores." The N-aminopeptidase receptor anchors the pre-pore in the lipid bilayer, pore formation affects integrity of the membrane,

**186**

*Mechanism of action of Cry proteins according to the sequential binding model.*

### **Figure 3.**

*Mechanism of action of Cry proteins according to the signaling pathway model.*

and electrophysiological evidence and biochemistry suggest that the pores cause an osmotic imbalance that causes cell death and lysis; the intestine is paralyzed, the insect stops feeding, and there is diarrhea, total paralysis, and finally death (**Figure 2**) [1, 29].

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].
