Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a gram-positive bacterium that has been used in the biological control of agricultural pests. It was named after the German city of Thuringia, where it was first isolated in 1911.

• Domain: Bacteria

• Phylum: Firmicutes

• Class: Bacilli

• Order: Bacillales

• Family: Bacillaceae

• Genre: Bacillus

• Species: Bacillus thuringiensis

Bacillus thuringiensis It has several subspecies and strains, each with specific characteristics that determine its effectiveness against different insect pests. Some of them:

• B. thuringiensis var. kurstaki: produces Cry proteins active against Lepidoptera caterpillars.

• B. thuringiensis var. israelensis: produces Cry and Cyt proteins effective against mosquito and simulid larvae.

• B. thuringiensis var. tenebrionis: active against beetles (Coleoptera).

• B. thuringiensis var. aizawai: used to control pests such as moths and caterpillars resistant to other strains of Bt.

Furthermore, each subspecies may present several strains, which differ in terms of efficacy, spectrum of action and ideal conditions of use.

Biology

Bacillus thuringiensis is a bacterium found naturally in soil, in aquatic environments, and even on leaves and plant surfaces.

Form resistant spores, which allow them to survive in adverse conditions, such as high temperatures and periods of drought.

During the sporulation process, Bt produces protein crystals known as Cry proteins or δ-endotoxins, responsible for its insecticidal action.

• Cry proteins: responsible for specific toxicity against insects. There are hundreds of variants of these proteins, each targeting different groups of insects (Lepidoptera, Diptera, Coleoptera, etc.).

• Cyt proteins: act as cytolysins, helping to destroy the epithelial cells in the intestines of target insects.

These crystals remain inert in the environment until they are ingested by susceptible insects. In the alkaline intestines of these insects, digestive enzymes activate Cry proteins, which then bind to the epithelial cells of the intestine, causing irreversible damage and leading to the death of the insect.

Bacillus thuringiensis consists of heterotrophic bacteria: it obtains energy and nutrients from the decomposition of organic matter present in soil, water and plant surfaces. It can grow in both aerobic and anaerobic conditions, although its ideal growth occurs in environments with available oxygen.

The Bt life cycle can be divided into two main phases:

• Vegetative phase: the bacteria is active and multiplies rapidly in nutrient-rich environments, such as decomposing organic substrates.

• Sporulation phase: When environmental conditions become unfavorable (such as lack of nutrients or water stress), Bt enters the sporulation phase. During this process, the bacteria synthesize protein crystals (Cry and Cyt proteins) and form extremely resistant endospores.

Spores can remain dormant in the environment for long periods, waiting for suitable conditions to germinate and restart the life cycle.

According to scientific studies, these proteins are highly selective, affecting only insects belonging to specific orders, such as Lepidoptera (caterpillars), Diptera (flies and mosquitoes) and Coleoptera (beetles).

Cry proteins

Cry proteins are crystalline and belong to the protoxin protein family. Synthesized in the inactive form (protoxin), they become toxic only after being processed in the intestine of target insects. Currently, more than 700 cry genes have been identified, classified into families and subclasses based on their molecular structure and specificity for different insect groups.

The major families of Cry proteins include:

• Cry1: active against Lepidoptera.

• Cry2: effective against Lepidoptera and Diptera.

• Cry3: targeting Coleoptera.

• Cry4 and Cry11: specific to mosquito and simulid larvae (Diptera).

The mechanism of action of Cry proteins involves several sequential steps:

• Insect ingestion: When a susceptible insect ingests leaves or substrates treated with Bt spores and crystals, Cry proteins are released into the intestine.

• Activation by digestive enzymes: In the alkaline environment of the insect gut, proteolytic enzymes (such as trypsin and chymotrypsin) cleave Cry protoxins, converting them into smaller, active toxins.

• Binding to intestinal receptors: the active toxins bind specifically to receptors present in the epithelial cells of the insect intestine. This binding is highly specific and depends on the presence of suitable receptors, which explains the selectivity of Cry proteins.

• Pore ​​formation and cell damage: After binding, the toxins insert themselves into the cell membrane, forming pores that allow the entry of ions and water. This causes osmotic imbalance, rupture of intestinal cells and death of the insect due to cessation of feeding, secondary infection or dehydration.

VIP Proteins

Unlike the famous Cry proteins, produced during the sporulation phase of the bacteria, Vip proteins are secreted continuously during the vegetative phase of growth, hence the name "Vegetative Insecticidal Proteins".

His discovery significantly expanded the biotechnological potential of Bacillus thuringiensis, offering new mechanisms of action and spectra of insecticidal activity that complement the Cry proteins already established on the market.

Vip proteins are organized into four main families, each with distinct structural features and specificities:

• Vip1 Family: comprises proteins with activity mainly against coleopteran insects (beetles) and some hemipterans. These proteins act as components of binary toxins, requiring the presence of Vip2 proteins to exert their full toxic activity.

• Vip2 Family: It functions as a complementary component of Vip1 proteins, forming binary toxic complexes. Although it does not have independent insecticidal activity, it is essential for the function of Vip1 proteins.

• Vip3 Family: This is the most studied and commercially relevant family, with specific activity against lepidopteran insects (caterpillars). Vip3 proteins act independently, not requiring partners to exert their insecticidal activity.

• Vip4 Family: most recently discovered family, still with few studies on its mechanism of action and potential application, but with indications of activity against Lepidoptera.

The mechanism of action of Vip proteins varies according to the family, but follows well-established general principles. For Vip3 proteins, which are the most widely used commercially, the process begins with the ingestion of the protein by the target insect during feeding.

In the insect midgut, Vip3 proteins are activated by specific proteases, undergoing cleavage that exposes active domains. These activated proteins bind specifically to receptors located on the membrane of midgut epithelial cells, including receptors such as cadherin, aminopeptidase N and alkaline phosphatase.

After binding to receptors, the proteins undergo conformational changes that allow them to be inserted into the cell membrane, forming oligomeric pores. These pores drastically alter the permeability of the membrane, causing osmotic imbalance, cell lysis and eventual paralysis of the digestive system. The insect stops feeding and dies from septicemia within 2-5 days.

One of the most valuable features of Vip proteins is their high specificity. Vip3 proteins, for example, are highly toxic to lepidopterans, including economically important pests such as:

• Spodoptera frugiperda

• Helicoverpa armigera

• Heliothis virescens

• Ostrinia nubilalis

• Diatraea saccharalis

This specificity is a result of co-evolution between Bt proteins and their target hosts, ensuring that non-target organisms, including beneficial insects, mammals and other vertebrates, are not affected.

Use for agricultural pest control

The use of Bacillus thuringiensis in the control of agricultural pests has revolutionized sustainable agriculture. It is widely used in the form of bioinsecticides, available both for direct application to crops and incorporated into genetically modified seeds (such as Bt varieties of corn and cotton).

Some of the uses:

• Caterpillar control: Bt is highly effective against caterpillars that attack crops such as soybeans, corn, cotton and vegetables. Cry proteins paralyze the digestive system of larvae, preventing them from feeding and causing their death.

• Bt seeds: In the case of crops such as soybeans, corn and cotton, genetically modified plants containing Bt genes produce Cry proteins, conferring resistance to pests such as the fall armyworm (Spodoptera frugiperda) and the boll weevil (Anthonomus grandis).