**3. Insect resistance management to the** *Bt* **technology**

The control of insect pests in agriculture has been done mainly through the application of chemical insecticides. However, chemical insecticides have lost effectiveness due to the selection of populations of resistant insects and cause adverse environmental effects.

In this context, the biological insecticide *B. thuringiensis* (*Bt*) has emerged as an alternative for the control of insect pests of agriculture. The Cry proteins produced by *Bt* have demonstrated a high specificity, and there is no evidence that directly affect natural enemies [15] as well as vertebrates [16]. These features have made the development of transgenic plants producing Cry proteins in its solubilized form possible, which give the property of resistance to insect pests. In the sequence, we will discourse about these proteins, as they are the mechanisms of action in the target insect, and their most important applications.

*Bt* is a gram-positive bacteria, strictly aerobic, which during its life cycle has two main stages: vegetative growth, which bacteria replicate by splitting, and sporulation, which is differenti‐ ating bacteria in the spore. *Bt* is considered a ubiquitous bacteria since it has been isolated from around the world in many different systems, such as soil, water, plant leaves, and dead insects, among others. In the sporulation phase, *Bt* bacterium is characterized by producing a para‐ sporal body known as "crystal," which is a protein nature and has insecticidal properties. The crystal protein is formed by proteins called δ-endotoxins, also known as Cry or Cyt proteins. δ-Endotoxin proteins have been found active against insects of Lepidoptera, Coleoptera, Diptera, Hymenoptera (ants), and also against other invertebrates such as nematodes, flatworms, and protozoans.

As mentioned, there are two types of δ-endotoxins: Cry and Cyt proteins. So far, more than 733 Cry genes and 38 different Cyt genes have been cloned and sequenced [17]. This is certainly a valuable arsenal for insect pest control. The nomenclature of δ-endotoxin is based solely on the similarity of the primary sequence. By definition, any parasporal protein that presents any toxic effect on body verified by bioassay or any protein that presents similarities with the Cry proteins are considered a Cry protein. Currently, Cry proteins have been found in other species of bacteria such as *Clostridium bifermentans* (classified as Cry16A and Cry17A) with activity to mosquitoes. The Cyt are *Bt* parasporal proteins that exhibit hemolytic activity.

Cry proteins are sorted and divided into 73 groups and several subgroups, and Cyt proteins into two different groups and subgroups, based on the similarity of the amino acid sequence. The Arabic numeral designates an identity of 45% (for example, Cry1, Cry2, etc.), the capital letter corresponds to 45–78% identity (cry1A, cry1B, etc.), the lowercase letter corresponds to the identities of 78–95% (Cry1Aa, Cry1Ab, Cry1Ac, etc.), and the Arabic numeral at the end of the nomenclature indicates more than 95% identity (Cry1Aa1, Cry1Aa2, etc.).

The symptoms observed in susceptible insect larvae when *Bt* crystals and spores are ingested are as follows: cessation of intake, intestinal paralysis, diarrhea, complete paralysis, and eventually death. In general, it is accepted that the Cry proteins are forming pores, which cause an osmotic imbalance in epithelial cells since proteins bind to receptors of the cell surface digestive system.

The Cry proteins are produced as a protoxin that needs to be proteolytically processed by proteases present in the gut of susceptible insects. This proteolytic processing releases toxic fragments to the insect (protein in the solubilized form), with a mass between 55 and 65 kDa, which interact with receptor proteins present in the microvilli of intestinal cells of the target insect. Subsequently, the proteins bind to the intestinal membrane forming a lytic pore.

Despite low similarity of Cry proteins, in some cases less than 25%, these have a similar structure composed of three domains. The domain I, composed of seven α and amphipathic antiparallel helices, where six of them surrounds the helix α5. This is the domain that forms the ion pore. Domain II consists of three folded β-sheet and three handles, where the most structural difference is observed. This is the domain less conserved among Cry proteins. However, its sequence and tertiary structure play an important role in the specificity of the protein since the handles interact with the receiver located in the microvilli of the midgut


epithelial cells. Domain III consists of two antiparallel β folded sheets forming a sandwich and is also involved in the interaction with receptors.

\*Insect resistant.

*Bt* is a gram-positive bacteria, strictly aerobic, which during its life cycle has two main stages: vegetative growth, which bacteria replicate by splitting, and sporulation, which is differenti‐ ating bacteria in the spore. *Bt* is considered a ubiquitous bacteria since it has been isolated from around the world in many different systems, such as soil, water, plant leaves, and dead insects, among others. In the sporulation phase, *Bt* bacterium is characterized by producing a para‐ sporal body known as "crystal," which is a protein nature and has insecticidal properties. The crystal protein is formed by proteins called δ-endotoxins, also known as Cry or Cyt proteins. δ-Endotoxin proteins have been found active against insects of Lepidoptera, Coleoptera, Diptera, Hymenoptera (ants), and also against other invertebrates such as nematodes,

As mentioned, there are two types of δ-endotoxins: Cry and Cyt proteins. So far, more than 733 Cry genes and 38 different Cyt genes have been cloned and sequenced [17]. This is certainly a valuable arsenal for insect pest control. The nomenclature of δ-endotoxin is based solely on the similarity of the primary sequence. By definition, any parasporal protein that presents any toxic effect on body verified by bioassay or any protein that presents similarities with the Cry proteins are considered a Cry protein. Currently, Cry proteins have been found in other species of bacteria such as *Clostridium bifermentans* (classified as Cry16A and Cry17A) with activity to

Cry proteins are sorted and divided into 73 groups and several subgroups, and Cyt proteins into two different groups and subgroups, based on the similarity of the amino acid sequence. The Arabic numeral designates an identity of 45% (for example, Cry1, Cry2, etc.), the capital letter corresponds to 45–78% identity (cry1A, cry1B, etc.), the lowercase letter corresponds to the identities of 78–95% (Cry1Aa, Cry1Ab, Cry1Ac, etc.), and the Arabic numeral at the end of

The symptoms observed in susceptible insect larvae when *Bt* crystals and spores are ingested are as follows: cessation of intake, intestinal paralysis, diarrhea, complete paralysis, and eventually death. In general, it is accepted that the Cry proteins are forming pores, which cause an osmotic imbalance in epithelial cells since proteins bind to receptors of the cell surface

The Cry proteins are produced as a protoxin that needs to be proteolytically processed by proteases present in the gut of susceptible insects. This proteolytic processing releases toxic fragments to the insect (protein in the solubilized form), with a mass between 55 and 65 kDa, which interact with receptor proteins present in the microvilli of intestinal cells of the target insect. Subsequently, the proteins bind to the intestinal membrane forming a lytic pore.

Despite low similarity of Cry proteins, in some cases less than 25%, these have a similar structure composed of three domains. The domain I, composed of seven α and amphipathic antiparallel helices, where six of them surrounds the helix α5. This is the domain that forms the ion pore. Domain II consists of three folded β-sheet and three handles, where the most structural difference is observed. This is the domain less conserved among Cry proteins. However, its sequence and tertiary structure play an important role in the specificity of the protein since the handles interact with the receiver located in the microvilli of the midgut

mosquitoes. The Cyt are *Bt* parasporal proteins that exhibit hemolytic activity.

the nomenclature indicates more than 95% identity (Cry1Aa1, Cry1Aa2, etc.).

flatworms, and protozoans.

68 Insecticides Resistance

digestive system.

\*\*Insect resistant and herbicide tolerant.

Source: CTNBio [20].

**Table 1.** General summary of maize plants genetically modified approved for marketing in Brazil.

The aminopeptidase N (APN) is a protein from the family of cadherins (BtR) and have been proposed as potential recipient of Cry1A proteins in Lepidoptera. The APN is a protein with an apparent mass of 120 kDa, which is anchored to the membrane via a glycosylphosphatidyl group inositol (GPI). There is evidence that the interaction of the protein with the cadherin receptor promotes an additional cut in the extreme amino terminus of the Cry protein by facilitating the formation of an oligomer or "pre-poro" formed by four monomers, which is responsible for membrane insertion and pore formation. For the "pre-poro" to be inserted in the membrane, it is necessary to interact with the APN receptor. The proteins anchored in the membrane by GPI are preferably distributed in specific regions of the membrane, known as lipid rafts, which have specific characteristics due to the high content of cholesterol and glycolipids. The interaction of the Cry protein of the "pre-pore" with the APN facilitates the insertion of oligomer in the lipid rafts on the membrane, resulting in pore formation [18].

The *Bt* technology relies on the transfer and expression of resistance genes to insect pest in maize, isolated from the bacteria *B. thuringiensis* Berlinger (*Bt*) [19]. The preservation of susceptibility to *Bt* toxins in pest populations depends on resistance management programs (IRM). Table 1 presents a summary of the most important technologies for maize crop.

#### **3.1. Considerations about the refuge area**

The main IRM strategy is the use of "high dose/refuge," which involves the use of high dose of *Bt* protein in plants, promoting high mortality of heterozygotes associated with the planting of refuge, i.e., a proportion of the crop in which it must be planted a non-*Bt* variety, allowing the survival of susceptible individuals to mate with possible resistant ones [21]. A protein may have high dose activity for a pest species and moderate or low dose to others, which does not impair the IRM because it is expected a simultaneous action of other mortality factors, such as natural enemies [22]. In this scenario, the adoption of the refuge area is also key to the IRM.

The explanation for cases of resistance to Bt crops appears to be related to the nonuse of high dose/refuge [23] strategy, particularly the nonadoption of refuge [24, 25].

The configuration of refuge areas may vary, but basic criteria of size and proximity to the *Bt* crops based on the target pest bioecology should be followed [22] so that these areas produce consistent proportions of adults for mating and maintaining susceptibility. In Figure 1, specified examples of refuge areas settings are shown.

**Figure 1.** Examples of refuge areas settings.

A good example of an alternative method for pest control, especially in Brazil, against fall armyworm, was the development and release of genetically modified plants such as *Bt* maize, a technology adopted with incredible rapidity in Brazil. Unfortunately, used without proper care, there are already complaints from different parts of the country in a few years of use about the presence of caterpillars and their damage above expectations. In fact, the expectation of farmers is that there would be no injuries from this pest in the crop. For fear of having economic losses, the chemical control so far left as low priority is back to be used in some areas of higher incidence even in *Bt* maize. Therefore, the importance of the technology should always be emphasized, however, pointing out that it alone will not solve the numerous phytosanitary problems in maize or other crops. For various reasons, since the commercial release of *Bt* maize, there was already a concern for the proper management of the technology to prevent breakdown of resistance by target pests. All good agricultural practices generally conveyed along with the acquisition of the seed must be strictly followed. Such practices include adopting refuge areas.

receptor promotes an additional cut in the extreme amino terminus of the Cry protein by facilitating the formation of an oligomer or "pre-poro" formed by four monomers, which is responsible for membrane insertion and pore formation. For the "pre-poro" to be inserted in the membrane, it is necessary to interact with the APN receptor. The proteins anchored in the membrane by GPI are preferably distributed in specific regions of the membrane, known as lipid rafts, which have specific characteristics due to the high content of cholesterol and glycolipids. The interaction of the Cry protein of the "pre-pore" with the APN facilitates the insertion of oligomer in the lipid rafts on the membrane, resulting in pore formation [18].

The *Bt* technology relies on the transfer and expression of resistance genes to insect pest in maize, isolated from the bacteria *B. thuringiensis* Berlinger (*Bt*) [19]. The preservation of susceptibility to *Bt* toxins in pest populations depends on resistance management programs (IRM). Table 1 presents a summary of the most important technologies for maize crop.

The main IRM strategy is the use of "high dose/refuge," which involves the use of high dose of *Bt* protein in plants, promoting high mortality of heterozygotes associated with the planting of refuge, i.e., a proportion of the crop in which it must be planted a non-*Bt* variety, allowing the survival of susceptible individuals to mate with possible resistant ones [21]. A protein may have high dose activity for a pest species and moderate or low dose to others, which does not impair the IRM because it is expected a simultaneous action of other mortality factors, such as natural enemies [22]. In this scenario, the adoption of the refuge area is also key to the IRM.

The explanation for cases of resistance to Bt crops appears to be related to the nonuse of high

The configuration of refuge areas may vary, but basic criteria of size and proximity to the *Bt* crops based on the target pest bioecology should be followed [22] so that these areas produce consistent proportions of adults for mating and maintaining susceptibility. In Figure 1,

dose/refuge [23] strategy, particularly the nonadoption of refuge [24, 25].

specified examples of refuge areas settings are shown.

**Figure 1.** Examples of refuge areas settings.

**3.1. Considerations about the refuge area**

70 Insecticides Resistance

Until 2007, the scenario of maize crop in Brazil was of growing losses by caterpillar's attacks. Problems with fall armyworm, black cutworm, corn earworm, cornstalk borer, and sugarcane borer increasingly frightened the farmer, who had little efficiency in the control of these pests using insecticides.

Quickly, the *Bt* technology in maize significantly reduced the problems with chewing insects, causing the erroneous impression that the technology was "bulletproof," meaning that nothing needed to be done and that all IPM practices could be left aside. However, with passing time and the intensive use of technology, the problems with insect resistance began to appear.

Resistance can be defined as a biological and evolutionary phenomenon that occurs in response to selection pressure exerted by the different control agents.

The evolution of resistance consists in the selection of resistant individuals that are naturally present in nature, leading to increased frequency of these individuals or their genes in the pest population, leading eventually to restrictions control agent efficiency. Unlike foliar insecti‐ cides, the *Bt* crops carry a much higher selection pressure on populations of insect pests that are target to control due to continued expression of insecticidal toxins over the crop growth period. This causes a higher risk of pest developing resistance to *Bt* technology.

The continuous expression of insecticidal proteins throughout the cycle of *Bt* plants and this rapid adoption represent threats to its durability, the strong selection pressure on the pest insects [23, 26]. Indeed, cases of resistance to *Bt* toxins have been reported for maize pests such as *S. frugiperda* [27–29] and *Diabrotica virgifera* [24].

According to Kumar et al. [30], the use of refuge areas, represented by planting susceptible varieties surrounding soybean crops sown with *Bt* varieties, is the main strategy to prevent the development of resistance.

Although avoiding the phenomenon of resistance of pests to insecticides (chemical or biolog‐ ical) should be a constant concern in the case of *Bt* crops, the recommended strategy involves actions that require time and use of machines, which may result in hatred of farmers to compliance, resulting in lower lifetime varieties with this feature.

The aggravating factor is that due to the characteristic (or genetic) of resistance when we start to see damage in the field in a technology with medium-high dose, the frequency of alleles is now probably around 10%, with the chance that, with continuous exposure to technology, the population will be at a much higher proportion of resistant individuals in a few generations.

Since the launch of the first *Bts*, several companies warned that the IPM practices should not be set aside and, especially, the refuge area should be established in all farms. The refuge, which is the planting of at least 10% of the area with a non-*Bt* hybrid maize, allows the survival of insects susceptible to *Bt* technology. The preservation of these susceptible insects allows the crossing with possible resistant insects, resulting in a progeny of susceptible insects.

However, few farmers planted the refuge area, and when they did, spraying in such areas were constant in order to obtain the productivity in the area. The result was that even when present, in many cases, the refuge areas did not work effectively in the maintenance of susceptible insects that would mate with any resistant insect coming from the *Bt* areas. In the absence of susceptible insects from the refuge area, any surviving insects resistant to exposure to *Bt* mated with each other, allowing relatively rapid increase of the resistance alleles and increased amounts of resistant individuals in the field.

Now that the resistance break of fall armyworm to Cry1F technologies is a reality, the question is, Is the refuge still necessary and beneficial for this technology?

The answer is certainly yes, because there are other pests that are controlled by the Cry1F protein as the sugarcane borer; other insects are likely to also develop resistance in case the best management practices are not applied, and in case there is no maintenance of susceptible individuals by adopting the structured refuge. The refuge is essential to maintain the efficiency of this control. In addition, all the technologies in the market today will have their increased durability and benefit from the adoption of best management practices and refuge areas for planting.

Knowing that the refuge areas are part of the IPM and the insect resistance management, how should we proceed to make the correct use?

As previously mentioned, poor adherence of refuge use or the many insecticide applications in the refuge, eliminating susceptible individuals, resulted in an ineffective resistance man‐ agement system, which favored a faster resistance evolution rate.

It is known that only the adoption of refuge is not enough to maintain the effectiveness of the technology and should also be considered to manage the use of insecticides in agriculture. The refuge should be as a donor area of susceptible insects so that they can mate with any resistant insects and the result is susceptible individuals in larger quanti‐ ties. Therefore, it is necessary to maintain differential applications between the refuge and *Bt* crop so that the application rate of insecticide in the refuge should be lower than in the fields. Basically, we have to think of resistance management in *Bt* area and management of economic damage in the refuge area.

### **3.2. Early desiccation followed by insecticide**

The aggravating factor is that due to the characteristic (or genetic) of resistance when we start to see damage in the field in a technology with medium-high dose, the frequency of alleles is now probably around 10%, with the chance that, with continuous exposure to technology, the population will be at a much higher proportion of resistant individuals in a few generations.

Since the launch of the first *Bts*, several companies warned that the IPM practices should not be set aside and, especially, the refuge area should be established in all farms. The refuge, which is the planting of at least 10% of the area with a non-*Bt* hybrid maize, allows the survival of insects susceptible to *Bt* technology. The preservation of these susceptible insects allows the

However, few farmers planted the refuge area, and when they did, spraying in such areas were constant in order to obtain the productivity in the area. The result was that even when present, in many cases, the refuge areas did not work effectively in the maintenance of susceptible insects that would mate with any resistant insect coming from the *Bt* areas. In the absence of susceptible insects from the refuge area, any surviving insects resistant to exposure to *Bt* mated with each other, allowing relatively rapid increase of the resistance alleles and increased

Now that the resistance break of fall armyworm to Cry1F technologies is a reality, the question

The answer is certainly yes, because there are other pests that are controlled by the Cry1F protein as the sugarcane borer; other insects are likely to also develop resistance in case the best management practices are not applied, and in case there is no maintenance of susceptible individuals by adopting the structured refuge. The refuge is essential to maintain the efficiency of this control. In addition, all the technologies in the market today will have their increased durability and benefit from the adoption of best management practices and refuge areas for

Knowing that the refuge areas are part of the IPM and the insect resistance management, how

As previously mentioned, poor adherence of refuge use or the many insecticide applications in the refuge, eliminating susceptible individuals, resulted in an ineffective resistance man‐

It is known that only the adoption of refuge is not enough to maintain the effectiveness of the technology and should also be considered to manage the use of insecticides in agriculture. The refuge should be as a donor area of susceptible insects so that they can mate with any resistant insects and the result is susceptible individuals in larger quanti‐ ties. Therefore, it is necessary to maintain differential applications between the refuge and *Bt* crop so that the application rate of insecticide in the refuge should be lower than in the fields. Basically, we have to think of resistance management in *Bt* area and management

crossing with possible resistant insects, resulting in a progeny of susceptible insects.

amounts of resistant individuals in the field.

should we proceed to make the correct use?

of economic damage in the refuge area.

planting.

72 Insecticides Resistance

is, Is the refuge still necessary and beneficial for this technology?

agement system, which favored a faster resistance evolution rate.

The previous crops as well as weeds and volunteer plants in the environment can host the main pests that attack maize in the initial phase, influencing the predominant species and the initial pressure of pests. Thus, in the no-tillage system, pest pressure in the early stage of the crop can be greater when compared to conventional tillage.

In the case of the presence of pests in the area, it is recommended that the application of insecticide be followed by preplanting desiccation, aiming the reduction of the initial popu‐ lation of pests, which are the most challenging for seed treatment; the control of resident caterpillars in later instars, which can cause early damage even in *Bt* maize crops; and the maintenance of the initial stand of the crop.

Regarding the early cover crop desiccation, it aims to provide dry straw on the ground, facilitating the operation of planting and promoting the protection of the soil. The optimal timing of herbicide applications may vary according to weather conditions and the cropping system used.

It is recommended to make two herbicide application; in the first period of approximately 30 days before planting, thus avoiding the presence of green mass at the time of sowing, and in the second desiccation shortly before planting in order to control the first flow of weeds after the first desiccation.

We highlight some benefits of desiccation performed at the right moment: more efficient use of insecticide in the second desiccation, as the green cover reduces its intensity with the first desiccation (eliminating the umbrella effect for insecticide); better plantability: easier cut of the straw by planter; availability of dry straw in the crop germination period: protection of soil moisture; reduction of possible allelopathic effects of the previous crop as the main crop; and ease in weed control in the postemergency phase of the crop, if necessary.

#### **3.3. Weed control**

Some weeds may host insect pests of succeeding crops, allowing a significant amount to survive in the areas of cultivation in the off-season period. In addition, weeds can be sources of caterpillars in later instars, which presents major difficulty to control by the *Bt* technology. Some practices may contribute to a better control of weeds, as well as prevent resistance to herbicides:


Regarding the management of volunteer plants after the maize crop, it is common the occurrence of germinação of remaining grains from previous crop spontaneously;

The amount and timing of germination of these maize kernels, producing crop residues (also known as "tigueras"), depends on many factors, being the quality of the previous harvest one of the most important; herbicides called graminicides are the main management tool of these plants. Volunteer plants are controlled until the V3/V4 stage to obtain consistent and quick controls. Weed competition is prevented with subsequent soybean crop, making the early management of volunteer plants.

#### **3.4. Seed treatment**

Seed treatment (ST) is a practice that seeks control of underground and initial culture pests, a period of great susceptibility to pests. The damage caused by these pests results in crop failures due to the attack on the seeds after planting, damage to roots after germination, and shoots of newly emerged plants. The correct choice of chemical is essential to the success of this operation. We recommend using products from broad spectrum to provide efficient control of the initial pests of the crop complex, which will bring results as the protection of plants in the initial development phase, broad-spectrum pest control, and maintenance of the initial stand of the crop.

#### **3.5. Crop rotation**

Crop rotation consists of alternating the planting of different species of crops in the same agricultural area. The choice of species for crop rotation should take into account economic factors, pests, diseases, and fertilization, among others.

To obtain maximum efficiency, improving productivity capacity of the soil, the planning of crop rotation must consider, preferably commercial plants and, whenever possible, involving species that produce large amounts of biomass and rapid development, cultivated singly or intercropped with commercial crops.

Among the benefits of crop rotation in pest management in *Bt* maize, the highlights are as follows: improved physical and chemical properties of the soil, reduction of disease inoculum source for subsequent crops, reduction of the initial population of some insect pests of the crop, aid in weed management, and ability to switch herbicides for the control and increase in the system productivity.
