Pest Control through Biological Contol Agenet

### **Chapter 2**

## Perspective Chapter: Secondary Metabolites of Entomopathogens as Biotechnological Tools for the Biological Control of Agricultural Insect Pests

*Hugo Rodríguez-Romero, Laura Rodríguez-Peláez, Adriana Reyes-Castro, Orlando Tlakaele Notario-Rendón, Manuel González-Peréz, Juventino Reza-Salgado, Pedro Antonio Rodríguez-Salazar and Jesús Antonio Salazar-Magallón*

#### **Abstract**

In recent years, the irrational application of chemical insecticides has caused the appearance of pest insect populations that are resistant to the active principles of commercial insecticides. In addition, these chemical compounds cause significant damage to the environment and to the people who apply them. The use of secondary metabolites produced by entomopathogenic microorganisms is a viable alternative that could mitigate the damage caused by chemical insecticides. Actually, the secondary metabolites of entomopathogens microorganisms have been studied; however, there are few reports on their massive production and their direct application as biological control agents. The aim of this book chapter is to describe, in a very general way, some of the secondary metabolites produced by entomopathogenic microorganisms, their potential application as bioinsecticides as well as their mass production.

**Keywords:** secondary metabolites, beauvericin, cry proteins, destruxins, biological control

#### **1. Introduction**

The presence of pest insect populations resistant to some chemical insecticides, caused by indiscriminate and irrational use, has produced enormous agricultural losses throughout the world. For example, a solution to combat the increase of pest insect populations has been to increase the recommended doses and the application times of the chemical insecticide, with consequent damage to the environment [1].

On the other hand, the impact on the exports of agricultural products is due to the restrictions given in the European Union and the United States regarding residual chemicals in vegetables and fruits [2].

Actually, an alternative to the use of chemical insecticides, is the application of entomopathogenic fungi and bacteria. These microorganisms have been used as biological control agents for pest insects since the beginning of the last century. For example, in Mexico, the use of Entomopathogenic Microorganisms (EM) has not become widespread, although the application of these microorganisms as part of plant health began more than 50 years ago; however, a significant increase in the use and commercialization of biological products have been observed since 1990 in all the world [3].

In fact, the reason for the increase in the use and applications of EM throughout the world has been because of its efficiency in killing insect pests, remaining long in the field after application, in addition to its specific interaction with the insect pest and be relatively safe in terms of the environment. On the other hand, the mechanisms of pathogenicity of EM have also been extensively studied and some of the compounds, called secondary metabolites, that participate in infectious processes have been described. This knowledge has allowed establishment of strategies to improve the production of secondary metabolites and their application as biological control agents [4].

The secondary metabolites are a group of compounds that have a vital role in infective and control processes. These compounds synthesized by EM do not play a direct role in growth or reproduction but rather have an adaptation function to the environment which surrounds them. Furthermore, they have their origin as derivatives of various intermediate compounds in primary metabolism. EM secrete a wide range of secondary metabolites that can be used in biological control [5].

For example, the secondary metabolites synthesized by Entomopathogenic Fungi (EF), such as oxalic acid*,* beauvericins, and dextrixins, which are toxic against insects. These compounds are produced when the fungus has penetrated the exoskeleton and has reached the hemocele, that is when it is considered that have insecticidal properties [6, 7]. Equally, some bacteria can produce proteins with insecticidal capacity, as is the case with the bacterium *Bacillus thuringiensis*. These proteins called Cry proteins and are capable of controlling insects, such as Lepidoptera, Coleoptera, and Diptera, among others. Certain virulence factors, such as VIP proteins, S-layer proteins, and some enzymes with insecticidal capacity, have also been described [8].

The aim of this book chapter is to describe the potential of the main secondary metabolites produced by entomopathogenic microorganisms, as biological control agents, as well as analyze the main routes and bioprocesses of biotechnological production of these compounds.

#### **2. Secondary metabolites of entomopathogenic fungi**

Currently, many secondary metabolites generated by different entomopathogenic fungi have been reported. Some secondary metabolites may be of simple organic structure, but regularly they are compounds of a slightly more complex structure. Furthermore, many secondary metabolites are cyclic and linear peptide toxins, which are derived from primary metabolites, and in some cases with unusual structures and occasionally accompanied by processes of specific biosynthesis [9, 10].

*Perspective Chapter: Secondary Metabolites of Entomopathogens as Biotechnological Tools... DOI: http://dx.doi.org/10.5772/intechopen.111465*

#### **2.1 Low molecular weight metabolites of entomopathogenic fungi**

In recent research, a considerable number of low molecular weight secondary metabolites have been reported, these compounds have been isolated from insect pathogens. **Figure 1** shows some secondary metabolites with insecticidal activity produced by entomopathogenic fungi. These metabolites have simple structures, such as oxalic acid, 2,6-pyridinedicarboxylic acid (dipicolinic acid), 4-hydroxymethylazoxybenzene-4-carboxylic acid. Some reports describe that the secondary metabolites of entomopathogenic fungi can alter the permeability of insect cell membranes, inducing the loss of fluids in the cells, they also modify the molting and metamorphosis process, change in fertility, and interferes with interactions. Ligand-receptor occur in the plasmatic membrane, deformations in the wings, and finally, cause the death of the insect [11].

#### *2.1.1 Oxalic acid*

The production of this secondary metabolite with insecticidal activity has been reported in *Beauveria* sp., *Lecanicillium (Verticillium) lecanii*, *Paecilomyces fumosoroseus* and *Metarhizium anisopliae*. This substance has been described as a virulence factor in phytopathogenic fungi, in addition to being studied as an element that participates in the solubilization of cuticular protein [12]. There are some reports where oxalic acid has been used to control *Varroa destructor*; this compound has been applied to the colony using the spray technique (obsolete method due to its complicated handling), by dripping and sublimation. All techniques have been highly effective (90–95% or more) in broodless colonies and therefore meet the requirements for a winter treatment [13]. According to Ref. [14], a solution of oxalic acid and ethanolwater in a ratio of 0.05, 0.1, 0.2, and 0.4 w/v; can be administered in amounts of 200 μL per treatment; in a single application and therefore meets the needs of the large-scale beekeeping industry.

#### *2.1.2 2,6-pyridinedicarboxylic acid*

This important compound (dipicolinic acid) has been produced by some entomopathogenic fungi, among which *Isaria* sp., *M. anisopliae, B. bassiana*, and *L (V) lecanii* [15, 16]. Its sodium salt has insecticidal properties against *Spodoptera frugiperda* larvae [16].

#### **Figure 1.**

*Structures of low molecular weight secondary metabolites. (a) Oxalic acid produced by Beauveria spp., Lecanicillium (Verticillium) lecanii, Paecilomyces fumosoroseus, and Metarhizium anisopliae. (b) Dipicolinic acid produced by Isaria spp., M. anisopliae, B. bassiana, and L (V). Lecanii.*

#### *2.1.3 4-hydroxymethylazoxybenzene-4-carboxylic acid*

It has been isolated together with its oxidation product (azoxybenzene-4,4-dicarboxylic acid) from the filtrate of the culture broth of *Entomophthora virulenta*, proving to be responsible for the insecticidal activity exhibited by this fungus against *Calliphora erythrocephala* [17].

#### **2.2 Toxins with a peptide nature**

Currently, cyclic and linear peptide toxins have been reported (**Figure 2**). The insecticidal action of these compounds has been described as very specific for certain groups of insects and their toxicity is due to the synergistic action of a complex group of compounds [18].

#### *2.2.1 Beauvericin*

Beauvericin has been the first molecule to be characterized due to its natural insecticidal properties, this compound was isolated for the first time from the mycelium of *Beauveria bassiana*, and in subsequent works, it has been extracted from different species of *Fusarium* and *Paecilomyces* species [18, 19]. Furthermore, beauvericin has been described as a cyclopeptide and its biosynthesis involves a multifunctional enzyme known as eniatin synthetase, whose expression is constitutive [18, 19]. In addition, it is known that this compound is an ionophore agent capable of forming Ca+ and K+ ion complexes that increase the natural and artificial permeability of membranes, inducing dehydration of tissues due to fluid loss from cells. Some reports indicate that this would be the main cause of the death of the insect, besides causing changes in the nucleus of the cells. This molecule has also been described and forms alterations in the molting and metamorphosis processes, as well as in fertility [19]. Some studies have reported that it has insecticidal properties against *Aedes aegypti* and *Hypothenemus hampei* mosquito larvae, producing paralysis after 6 hours of treatment and 73% mortality at 72 hours [20].

#### *2.2.2 Efrapeptins*

These molecules constitute a highly varied mixture of antibiotic peptides generated by some fungi, such as *Tolypocladium niveun*, *Beauveria nivea*, and *Tolypocladium* 

#### **Figure 2.**

*Chemical structures of some toxins of a peptide nature. (a) Beauvericin, produced by Beauveria bassiana and Paecilomyces sp. (b) Efrapeptin F generated by Tolipocladium niveun, Beauveria nivea, and Tolipocladium cylindrosporum. (c) Destruxin a produced by Metarrizhium anisopliae.*

*Perspective Chapter: Secondary Metabolites of Entomopathogens as Biotechnological Tools... DOI: http://dx.doi.org/10.5772/intechopen.111465*

*cylindrosporum* [21]. A nonpolar extract from a culture broth of *Beauveria bassiana* showed toxic activity against the red potato beetle. In fact, this extract, after appropriate fractionation, resulted in a peptide fraction identified as a mixture of efrapeptins, which was isolated by HPLC in five different peaks. These peaks were purified and characterized chromatographically, turning out to be rich in alpha-aminoisobutyric amino acid, and contained at least 15 other amino acid residues, in addition to having the acetylated N-terminus [22]. Efrapeptins produced by T*. cylindrosporum* have been shown to interfere with agglutinin in *Galleria mellonella* larvae, suggesting that these metabolites may interfere with ligand-receptor interactions that occur at the plasma membrane, allowing us to infer that most interactions between cells and humoral components of the insect immune system are receptor-mediated [23].

#### *2.2.3 Destruxins*

They are the best-characterized compounds since their mode of action also inhibits DNA, RNA, and protein synthesis in insect cells. One of the first studies aimed at the detection of fungal toxic substances has been on *Metarhizium anisopliae* strains, leading to the isolation of two cyclic depsipeptides that were called destruxins A and B. Today, 14 species of destruxins with variable insecticidal activities are known. This type of toxin has been analyzed and is capable of inhibiting the secretion of fluids through the Malpighi tube in *Schistocerca gregaria* [24]. The destruxins A, B, and E that have been produced by *M. anisopliae*, exhibited insecticidal properties when tested in *Plutella xylostella* larvae, with a high level of mortality in insect populations. They also caused deformations in the elytra and forewing of the insect. The influence of destruxin E on calcium flux and protein phosphorylation has also been observed *in vitro* cultures of Lepidoptera [25].

On the one hand, bassiacridin is a toxic protein, it has been purified from a strain of Beauveria bassiana by chromatographic methods. In a recent study, bassiacridin showed no affinity for anion exchangers and was characterized as a 60 KD monomer and an isoelectric point of 9.5. Furthermore, this molecule was shown to have β-glucosidase, N-galactosidase, and N-acetylglucosaminidase activity, as well as a proven insecticidal action [26].

#### **3.** *B. thuringiensis* **secondary metabolites**

δ-endotoxins produced particularly by *B. thuringiensis* (Bt) are parasporal protein bodies made up of polypeptide units of different molecular weights, from 27 to 140 kDa. Currently, 300 holotypes of *B. thuringiensis* toxins have been reported, classifying them into 73 Cry and 3 Cyt families. Bt toxins are produced during the sporulation phase, the Cry (crystal) protein is known for its specific toxic effects on a target organism (most belong to the order of insects), likewise the Cyt (cytolytic) proteins have been related with toxic effects on a wide variety of insects, mainly Diptera; however, its cytotoxicity against mammalian cells has also been proven [27].

Furthermore, during the vegetative cycle, *B. thuringiensis* produces soluble virulence factors of various types; some researchers have proposed that they be considered additional virulence factors to δ-endotoxins, which would explain the high pathogenicity of this bacterium toward its target organisms. In addition, some of these alternative virulence factors include -β-Exotoxins (Thuringiensins), nucleotide analogs of ATP and UTP that are synthesized by enzymes whose genes are found on

conjugative plasmids, compete for RNA polymerases and thus block rRNA synthesis in mammals; by inhibiting transcription, they exert mutagenic activity. They are used for house fly control in Africa. VIP proteins (Vegetative Insecticidal Proteins), these proteins have crystallized and contain a domain similar to the active site of proteins with ADP-ribosylation activity SIP proteins [28].

#### **3.1 Cry proteins**

Cry toxins were the first described proteins in *B. thuringiensis*, being the most diverse group, with 78 different protein families and a total of 817 described proteins. Cry proteins are active against a wide range of insect species (Lepidoptera, Coleoptera, Aphids, Nematodes, etc.). The continuous and growing discovery of a wide variety of Cry toxins then demanded the creation of a nomenclature system that would allow them to be classified in an orderly manner. The classification system for these toxins was designed and proposed by the *B. thuringiensis* Toxin Nomenclature Committee, which was based on the identity percentage of the amino acid sequence of each protein. In this way, when a new *B. thuringiensis* toxin is discovered, it is assigned a name by comparing it with its closest counterpart [29].

Additionally, the mechanism of action of Cry proteins was mainly described in Lepidoptera as a multistep process. *B. thuringiensis* crystals are ingested and then solubilized in the insect's midgut, after which the crystal proteins are released as protoxins. These will not cause damage *per se* but must be processed by intestinal proteases to generate active toxins that will lead to the death of the larva. In their monomeric form, the toxins cross the peritrophic membrane and bind univalent to cadherin, with great affinity for the apical face of the epithelial membrane. Then, according to studies carried out in insect cell cultures, a magnesium ion-dependent signaling cascade is initiated that would be responsible for cell death [30].

Furthermore, the initiation of this signaling cascade stimulates the exocytosis of cadherin from intracellular vesicles to the apical membrane of the cell and increases the number of receptors; therefore, it recruits a greater number of free toxins that would amplify the initial signal. On the other hand, based on *in vitro* experiments and bioassays, it is proposed that the binding of monomers to cadherin facilitates proteolytic cleavage on the N-terminus of the toxin. This last cleavage induces the assembly of the monomers and a pre-pore oligomeric form is established. This structure increases the affinity for a secondary receptor, such as aminopeptidase N or alkaline phosphatase. Finally, binding to this second receptor facilitates the formation of a pore in the epithelium of the midgut, which causes an osmotic imbalance and the consequent cell lysis. The intestinal tissue is seriously damaged, which prevents the assimilation and retention of vital compounds for the larva and leads to the death of the insect. Death can be accelerated by germinating spores and proliferating vegetative cells in the insect's hemocoel [31].

Due to the success in biological control, some brands have developed commercial products based on *B. thuringiensis*. For example, The Abbott Company has, among others, products based on the kurstaki serovars like Dipel (cry1Aa, cry1Ab1, cry1Ac1, cry2Aa1, cry2Ab1 genes) and Xentari (*B. thuringiensis* aizawai with cry1Aa1, cry1Ab1, cry1Ba1, cry1Ca1, cry1Da1 genes) that they are lethal against lepidopteran insects. Ecogen Inc. has promoted the biopesticides Lepinox (based on *B. thuringiensis* kurstaki) and Crymax (*B. thuringiensis* strain ED7826 with cry1Ac, cry2A, and cry1C genes) for lepidopteran lethality. Biochem has developed a formula based on *B. thuringiensis* israelensis for the control of Diptera (Cry4Aa1, Cry4Ba1,

Cry10Aa1, Cry11Aa1 toxins). Mycogen developed M-trak, a product for which it was sued by the company Novo Nordisk for misuse of the strain [32].

#### **3.2 Cyt proteins**

Cyt proteins have been found in the parasporal crystal produced by *B. thuringiensis* and they are active against Diptera. These proteins present cytolytic activity *in vitro* assays so that Cyt proteins break intestine cells due to their cytolytic activity and not by forming pores like Cry proteins. So far, known members of the Cyt proteins include the Cyt1, Cyt2, and Cyt3 protein families. Cyt proteins have a molecular weight of 28 kDa, which is a cytolysin of nonspecific action, produced mainly by the israelensis variety, accumulating in the crystal, together with the endotoxins typical of this variety. Because they have no homology to the other Cry proteins, the Cyt toxins are not classified as endotoxins. The general structure of Cyt proteins has an α/β-like domain with a β-sheet in the center surrounded by two α-helicals. The central β-sheet consists of six antiparallel β-strands, flanked by a layer of α-helices [33].

According to the studies of Ref. [34], the amplification of fragments of the expected size was obtained by PCR using pairs of oligonucleotide primers that detect the genes cry4Aa, cry4Ba, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, and cyt2Aa. The analyzes confirmed that, of the 1073 isolates subjected to PCR, only 45 (4.2% of the total) presented amplification for a single gene or the combination of them among some isolates. Of the 45 isolates (specific to Diptera) of *B. thuringiensis* subjected to selective bioassays, 13 (28.9%) presented 100% mortality against *A. aegypti* larvae, which were subjected to quantitative bioassays for the calculation of LC50 and LC90.

The relationship between toxicity and gene content of the *B. thuringiensis* isolates studied suggests a joint action of the toxins in the control of *A. aegypti*. Isolates carrying three or more Diptera-specific genes (cry and cyt) were effective in selective bioassay tests, causing 100% mortality, which is equivalent to treatment with the standard strain *B. thuringiensis* var. israelensis. Some isolates showed three combinations of genes and showed no control efficacy against insect larvae. In these combinations, the cry4Aa and cry4Ba genes were not present and, in isolates 574 and 582, the cry11Aa gene was not present either [33, 34].

In studies carried out by [35], the strains LBT-63 and LBT-87 have shown amorphous crystals very similar to those found in *B. thuringiensis* subs. Israelensis; however, strains LBT-62 and LBT-99 showed very small inclusions attached to the spores. The evaluation of the biological activity of strains LBT −62 and LBT 83 and LBT-99 against *D. melanogaster* have shown how the LBT-87 strain has a 70% mortality in the larvae. Of the larvae that managed to survive and form pupae, they never reached the adult stage. The remaining strains only slightly delayed pupal formation, but adult emergence eventually occurred. Although the LBT-87 strain showed an effect against *D. melanogaster*, the virulence was lower than that obtained with the standard strain *B. thuringiensis* subs. Israelensis, specific for Diptera.

#### **3.3 VIP proteins**

VIP proteins (Vegetative Insecticidal Proteins) constitute another family of insecticidal proteins produced by some strains of *B. thuringiensis*. Such proteins do not resemble Cry proteins and are produced during the vegetative phase of the bacterium, in addition to being soluble, do not form crystals and have the same magnitude of toxicity as Cry proteins. In addition, the insecticidal spectrum of VIP proteins includes

Coleoptera (VIP1 and VIP2) and Lepidoptera (Vip3), including some species of insect pests that are insensitive to the action of Cry proteins. The *Vip2A(a) and Vip1A(a)* genes of some *B. thuringiensis* strains are known to be located in plasmids. Also, the proteins encoded by these genes contain a signal peptide typical of secretory proteins in the N-terminal region. In addition, it is known that both Cry and VIP proteins, toxic to beetle larvae, have similar sequences and probably a similar mode of action, since both are based on binding to specific membrane receptors of midgut epithelial cells of the target insects and in the consequent formation of pores [36].

Through histopathological observations, it was possible to verify that the epithelial cells of the midgut of susceptible insects are the main target of the insecticidal protein VIP3A, which causes intestinal paralysis, complete cell lysis, and the consequent death of the larvae. Thus, disruption of intestinal cells appears to be the main mechanism for the lethality of VIP proteins. In addition, the production of the VIP3A protein by vegetative cells after spore germination is an important factor in combined spore toxicity in insect species in which the Cry proteins are relatively inactive [37].

#### **3.4 S-layer proteins**

The surface layers (S-layers) proteins are known as monomolecular crystalline arrays of proteinaceous subunits, these compounds are over the surfaces of many bacteria and archaea [38]. Besides, S-layer proteins frequently have demonstrated a big capacity for self-assembly during their formative stage. Several signal peptides and three S-layer homology (SLH) domains, which are anchored to the cell surface are found at the N-terminal part of S-layer proteins and various S-layer genes from *B. thuringiensis* have been cloned and sequenced [39]. Among these *B. thuringiensis* strains, only one S-layer of *B. thuringiensis* subsp. finitimus strain CTC has been thoroughly studied for its biochemical characterization [40].

According to Ref. [41], four *B. thuringiensis* strains whose parasporal inclusions contained the S-layer protein (SLP) and cloned two slp genes from each strain. So, phylogenetic analysis indicated these SLPs could be divided into two groups, SLP1s and SLP2s. To confirm whether SLPs were present in the S-layer or as a parasporal inclusion, strains CTC and BMB1152 were chosen for further study. Western blots with isolated S-layer proteins from strains CTC and BMB1152 in the vegetative phase showed that SLP1s and SLP2s were constituents of the S-layer.

On the other hand, Ref. [42] reported the identification of an S-layer protein by the screening of *B. thuringiensis* strains for activity against various insect pests as the coleopteran *Epilachna varivestis* (Mexican bean beetle; Coleoptera: Coccinellidae). Some of the *B. thuringiensis* strains assayed against *E. varivestis* showed moderate toxicity. However, a *B. thuringiensis* strain (GP1) that was isolated from a dead insect showed remarkably high insecticidal activity. The parasporal crystal produced by the GP1 strain was purified and shown to have insecticidal activity against *E. varivestis* but not against the lepidopteran *Manduca sexta* or *Spodoptera frugiperda* or the dipteran *A. aegypti*.

#### **4. Development and escalation in the production of secondary metabolites**

In general lines, the development of biopesticides has a close relationship with the study and exploitation of modern biotechnology, therefore, so that the entities related to such initiatives (industry, universities, and research centers, among others) can

#### *Perspective Chapter: Secondary Metabolites of Entomopathogens as Biotechnological Tools... DOI: http://dx.doi.org/10.5772/intechopen.111465*

generate innovation and technological development requires a highly qualified human resource of a multidisciplinary nature [43]. In this context, technological development can be understood as the use of existing scientific knowledge for the production of new materials, devices, products, procedures, systems, or services, as well as for their substantial improvement, which includes prototyping and pilot installations [44]. However, the technological development of a microbial biopesticide is a complex task that involves not only technical-scientific stages but also stages associated with the analysis of economic viability and market potential, as well as compliance with national regulations and regulations for bioproducts [45].

Other important aspects in the development of a biopesticide are the costs of implementation and execution, the relative complexity, and the duration of each stage. Samada et al. [46] have described as technological development progresses, possible microorganisms or candidate isolates are screened, but, in turn, the costs, complexity, and time required increase due to multiple tests, bioassays, analysis, and studies that must be carried out to ensure the efficiency and reproducibility of the bioproduct under controlled, semi-controlled, and field conditions. Once all the stages of the development of a biopesticide have been executed (which can take between 4 and 5 years), it can be ensured that the technological development is efficient, effective, and stable, which allows projecting the commercialization and distribution of the biopesticide in the emerging market of bioproducts worldwide.

The need for sustainable development of agriculture has fostered many initiatives that have stimulated the development of alternative methods that reduce the use of chemical pesticides in pest control. Microbial biopesticides, therefore, represent one of the most promising alternatives and, although their commercialization remains marginal, the demand is constantly increasing in all parts of the world. This significant increase coincides with the growth of biological pest control in high-value crops, such as greenhouse-produced vegetables, fruit crops, vineyards, and forestry, among others. On the other hand, despite the fact that biological control has shown an important development in organic agriculture, the most promising future of biopesticides is found in integrated pest management (IPM) programs [47].

Even though there are significant advances in scientific and technological knowledge on the development of biopesticides, there are few cases in which formulations with a high and consistent biocontrol activity have been produced, which also have a wide spectrum of use and respond to the challenges cheaply. Although many bioproducts have been developed, several of them have been withdrawn from the market before achieving commercial success. In recent years, there have been important advances in the development and industrial production of biopesticides, but they still occupy a small percentage of the products used for crop protection. In many cases, research centers or companies develop excellent biopesticides but fail to position them before producers. For this reason, a product is only effective if it generates impact in field conditions and if it is supported by a solid market strategy that guarantees reliability and profitability [48].

#### **5. Conclusions and perspectives**

The production of a biopesticide not only requires detailed knowledge of the microbiology and physiology of the biocontrol microorganism but also knowledge of the biology of the pest, the epidemiological aspects that define its harmful effect on the crop, as well as the physiology of the pest. Plant. In addition to this, several

technological challenges related to the fermentation process, the type of formulation, the pest population versus the biocontrol microorganism population, and the application systems used must be addressed. Therefore, before the development and application of a formulation, it is necessary to understand the ecology of the interaction between the biocontrol microorganism, the host plant, and the pest. Likewise, in the development of biopesticides, each step must be considered: the selection of the microorganism, the production method, the delivery system, the application technology, the factors that affect its development and persistence in the environment and, ultimately, instance, the availability of the product in the market and the various positioning actions of this to achieve recognition and acceptance by farmers.

#### **Acknowledgements**

We thank the Faculty of Sustainable and Protected Agriculture, as well as the academic team in sustainable and protected agriculture, for the support provided during the completion of this study.

### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Hugo Rodríguez-Romero, Laura Rodríguez-Peláez, Adriana Reyes-Castro, Orlando Tlakaele Notario-Rendón, Manuel González-Peréz, Juventino Reza-Salgado, Pedro Antonio Rodríguez-Salazar and Jesús Antonio Salazar-Magallón\* Faculty of Engineering in Sustainable and Protected Agriculture, Agricultural Bioengineering Laboratory, Avenida Universidad Tecnológica #1, México

\*Address all correspondence to: biologicalcontrollab@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Secondary Metabolites of Entomopathogens as Biotechnological Tools... DOI: http://dx.doi.org/10.5772/intechopen.111465*

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#### **Chapter 3**

## Perspective Chapter: Pheromone-Based Techniques in Sustainable Pest Management

*Melis Yalçın*

#### **Abstract**

The intensive use of insecticides can result in environmental pollution and adverse effects on human health due to the issue of insecticide residue in the environment. To mitigate this, various control techniques, including cultural, biological, and biotechnical methods, or their combinations, can be employed to manage invasive species. One such biotechnical method that has become popular is the use of pheromones. Pheromone techniques enable early pest detection, population monitoring, mass trapping or annihilation, and mating disruption. To maximize their effectiveness, it is important to determine the exact pheromone component, optimization rate, trap design, and saturation rate for each species. In conclusion, implementing these different pheromone-based strategies is essential for providing effective pest management strategies that take regional variations in pheromones into account.

**Keywords:** pheromone, pests, monitoring, mating disruption, pest management

#### **1. Introduction**

Pheromones that represent intraspecific communication of the species are considered environmentally friendly for monitoring and controlling insect species. Chemical communication leads to emitting species-specific signals of individuals which causes reactions in different life forms of the same species. In the past pheromones were described as; chemicals involved in intraspecific communication which describes the term "ectohormone" [1] but the term changed into pheromone in 1959 [2, 3]. Detection of the first sex pheromone of the silkworm moth, *Bombyx mori* (Lepidoptera: Bombycidae) has revealed the necessity of studies on pheromones [4]. In the last decades, more than 600 species of Lepidopteran pheromones were discovered [5, 6] as a consequence sex pheromone global market size increased to 2.4 billion USD per year in 2019 [7]. After World War II, the usage of broad-spectrum insecticides increased dramatically, leading to intensive usage of chemicals which in turn caused residues on food, development of pest resistance, environmental pollution, and posed risks to the health of humans and other living organisms [8]. In the past decades, there are lots of studies about pheromone biosynthesis, new insect pheromones, their modes of action, and their application in integrated pest management.

Integrated pest management approach combined with chemical ecology, insect behavior and interaction between organisms provides exploring the pheromones of pests [9]. Pheromones are successful when the pest population density is low, they provide long-term reduction in pest populations and generally do not affect natural enemies.

Insect sex pheromones comprise different components usually one chemical is primary component and efficient to attract mates [10, 11]. Based on their chemical structure and biosynthetic origin, various types of pheromones exist. Type I pheromones, for instance, are characterized by the presence of acetates, alcohols, and aldehydes, and constitute approximately 75% of all moth sex pheromones that are currently known [12]. Type II pheromones consist of hydrocarbons and epoxide derivatives with carbon atoms [12] and contain about 15% of moth sex pheromones reported. Type III pheromones contain hydrocarbons that involve methyl branches. Type 0 pheromones involve methyl-carbinols and methyl ketones and generally belong to the Eriocraniidae and caddisflies (Tricoptera) [6, 13]. The chemical structure of pheromones comprises of carboxylic acids, hydrocarbons, epoxides, lactones, alcohols, esters, aldehydes, ketones, isoprenoids, and triacyl glycerides [6, 14, 15].

There are different kinds of pheromones in the nature. Releaser pheromones such as alarm pheromones cause sudden changes in the behavior of pests while primer pheromones cause slower and longer physiological changes [16–18]. In the presence of the menace, the secretion begins between the alert members of the same species. After sex pheromones, the alarm pheromones are the most common pheromone produced by insects. Generally, alarm pheromones of some aphid species involve germacrene A, α-pinene, and sesquiterpene (E)-β-farnesene (EBF) [19, 20]. Alarm pheromones are released by some insects to avoid attack by natural enemies. Avoidance and dispersal behavior can be observed when alarm pheromones are released. But some social insects such as bees and ants may respond aggressively to alarm pheromones [16]. Sex pheromones generally attract males over long distances and very low concentrations of sex pheromones can be detected by sensilla of insects [21]. Studies concerned about sex pheromones consist of Lepidoptera, Diptera, Hemiptera, Coleoptera, and Blattodea since 2000. Especially Noctuidae, Tortricidae, Plutellidae, Crambidae, Bombycidae, Pyralidae, Lymantriidae, Gelechiidae, Geometridae, Lasiocampidae, Sesiidae, Gracillaridae, Erebidae family from Lepidoptera order, Drosophilidae, Tephritidae, Psychodidae from Diptera order, Pseudococcidae and Miridae from Hemiptera order, Scarabaeidae and Buprestidae from Coleoptera order, Blatteidae from Blattodea order were the most studied ones. EPA gave permission in 1978 for the registration of Gossyplure which was the first registered product used for the management of pink bollworm [22]. Insect sex pheromones generally consist of more than three double bonds, aliphatics of 9–18 carbons, and end with an acetate, aldehyde, or alcohol [20]. The weather conditions, time, and host plant availability are important for the release of sex pheromones [17]. Aggregation pheromones provide to generate groups and mating [23] and some species of bark beetles (Scoliytidae: Coleoptera) release aggregation pheromones for mating and feeding [24]. Conversely, anti-aggregation pheromones cause dispersal of individuals for both sexes to find optimum space to feed. Ovipositiondeterring pheromones warn insect species to avoid egg-laying on hosts such as females of fruit flies (Diptera: Tephritidae) *Ceratitis capitata* release oviposition determining fruit making pheromone after egg-laying [25]. Trail pheromones provide insects to find nest sites and to mark feeding for social insects such as termites and ants [18]. Home recognition pheromones are observed in social insect colonies. Queens of social insects release "Queens pheromones" to attract workers. Queens benefit for her care and protection while workers gain information about their queen [20]. There are also recruitment

pheromones and royal pheromones. Recruitment pheromones lead to leave the nest and migrate to work. Terrestrial ants use recruitment pheromones to follow the trial. Royal pheromones provide termites to find the queen and attract them to follow the trial [20].

#### **2. Pheromones with plant volatiles**

Pheromones and pheromones plus plant volatiles and pests respond to those molecules and is gaining importance [26]. The effectiveness of pheromones can be enhanced by plant volatiles (**Table 1**). In one of the experiments it was mentioned that traps baited with pheromones and host plant, volatiles captured more adults than traps pheromone alone [28]. Aggregation of pheromone ferrugineol with plant volatiles was more effective for the control of *Rhynchophorus ferrugineus* (Coleoptera: Curculionidae) [29]. Green leaf volatiles (GLV) are six carbon atom groups and consist of alcohols, aldehydes, and acetones. They can improve or inhibit the response of insects to their pheromones. For instance, boll weevil aggregation pheromone with GLV-2-hexenol led to more weevils caught in traps in contrast to pheromone alone [34, 35]. Another example of boll weevil traps comprised of aggregation pheromone of *Anthonomus grandis* with green leaf volatiles from cotton plants [30]. Fruit volatile benzaldehyde with plum curculio aggregation pheromone increased the trap captures [31, 36]. Similarly, grandisoic acid which is an aggregation pheromone used for monitoring and control of the plum curculio in apple trees [37]. Deciduous plants volatile with pheromones of corn earworm and the codling moth were also more effective than pheromone alone [27]. But GLVs with pheromones interrupted pheromone responses in the *Tomicus piniperda*, *Conophthorus resinosae, Dendroctonus pseudotsugae,* and *Ips typographus* [34]. (Z)-3-hexenyl acetate is an important chemical to locate the diamondback moth *P. xylostella*'s host [38]. This compound coupled with the synthetic


#### **Table 1.**

*Experiments about pheromone and host volatiles.*

pheromone resulted the highest response of moths. Antennal receptors show that some olfactory receptor neurons (ORNs) responded to GLVs besides GLV's synergized responses of ORN [39]. Under aphid attack, plants release some compounds which trigger the chemical defense mechanism. These compound attract the aphid predators. In the presence of methyl salicylate the colonization of the aphids was reduced. Methyl salicylate also inhibits males of the *Pieris napi* from mating.

### **3. Trap design**

There are some important properties for trap design. These are height, size, shape, alignment at right angles to the wind, position, and timing of the trap [20]. Some weevils preferred light red color for traps. For efficient usage, traps should be at an exact height such as 50 cm above from the ground [40]. **Figure 1** illustrates four different pheromone baited traps for monitoring Pea Leaf Weevil. Pheromone-baited pitfall and ramp traps caught more adults than delta and ground traps. In both pea and lentils, trap location, type of dispenser, dose, and purity of the pheromone, and environmental conditions will affect the trap catches achievements [41].

In one of the monitoring experiments, the funnel traps were more attractive when compared to adhesive traps for the monitoring of *Palpita unionalis* Hubner (Lepidoptera: Pyralidae) and also more males were caught in the edge instead of the interior part of the forest [42]. Identification, count and removal were easier than adhesive traps and also contamination was less in the funnel trap. It is also mentioned that traps for *Cydia pomonella* located along the border of the orchards were more successful when compared to traps located 30–50 m inside [43]. Location of the trap such as in apple trees traps positioned at 4 m captured more *Cydia pomonella* L.

#### **Figure 1.** *Different trap types for Sitona lineatus: (a) Delta Trap, (b) Ground Trap, (c) Ramp Trap, (d) Pitfall Trap [41].*

#### *Perspective Chapter: Pheromone-Based Techniques in Sustainable Pest Management DOI: http://dx.doi.org/10.5772/intechopen.110862*

(Lepidopetra: Tortricidae) male compared to those positioned 2 m [44]. Also in one of the experiments traps placed on the top of the tree captured more *Choristoneura rosaceana* Harris (Lepidopetra: Tortricidae) than traps placed in the middle and at the end [45]. One of the experiment groups tested three categories of traps that are bucket style trap, sticky trap, and local trap with three pheromone lure type, they are four-component lure (4C), two-component lure, and three-component lure (3C) for the management of *Spodoptera frugiperda*. In conclusion, bucket-style trap with 3C lures attracted more fall armyworm moths than other combinations with trap and lures. Another experiment concerning trap design for catching *Anoplophora glabripennis* (Coleoptera Cerambycidae) [46]. For monitoring intercept panel traps hung on poplar trees, screen sleeve traps wrapped around poplar trunks, intercept panel traps hung on bamboo poles 20 m away from host trees were used. Male produced pheromone alone and baited with a mixture of (−)-linalool, (Z)-3-hexen-1-ol, linalool oxide, trans-caryophyllene, and trans-pinocarveol. Screen sleeve traps baited with a combination of (−)-linalool and the pheromone caught the highest number of beetles while bamboo poles hung on poplar trees caught the lowest number [47]. It is also mentioned that optimization of pheromone lures and the trap design for monitoring *Dioryctria abietivorella* (Lepidoptera: Pyralidae) is essential for the management of this pest [48]. Three types of sticky trap (White diamond trap, green delta trap, white wing trap) and a green bucket type trap were compared and the most successful trap was found diamond trap while the least males bucket-style bucket style white trap. There are two types of pheromone application methods. One of them is using pheromones directly which includes mass trapping and area-wide dissemination applications. Area-wide dissemination includes mating disruption, attract & kill, and push & pull strategy. The other one is using pheromones indirectly includes monitoring and detecting the spraying time strategy.

#### **4. Insect pheromones with different application techniques**

#### **4.1 Population monitoring**

This management technique is responsible for maintaining various aspects such as species detection, controlling treatment time, targeting pest dispersion, early warning, and population density and fluctuation, based on the specific needs of the situation. [49, 50]. At the same time, biodiversity hotspots can be identified and species-specific pests can be monitored without risks to non-target organisms with this management strategy [51]. Synthetically derived pheromone was formula into the dispenser and trap to attract the pests. There are different kinds of pheromone dispensers for monitoring insects population such as plastic laminates, twist ties, wax formulations, polyethylene vials, rubber septa, hollow fibers, and impregnated ropes. The benefits of pheromone-based monitoring include affordability, specificity to certain species, and user-friendliness. However, the success of the process also depends on crucial factors such as the rate of pheromone release, the trap's design, its color, and the strategic placement of the traps [42, 48, 52–59]. In the population monitoring technique, to monitor *Conotrachelus nenuphar* (Coleoptera: Curculionidae) apple trees were baited with grandisonic acid (aggregation pheromone) and benzaldehyde (synthetic fruit volatile) to attract curculio adults. Sex pheromones produced by females are used for the monitoring of *Lymantria dispar* L. (Lepidoptera: Lymantriidae) [60], *Heliothis* spp. (Lepidoptera: Noctuidae) populations [61] and Codling moth [62].

Aggregation pheromones usually secreted from males and attract both sexes of pest to mate and feed. They are used to monitor the boll weevil and plum curculio [63–66]. Some of the pheromone compounds, their hosts, and invasive species were given in **Table 2**. But these pheromone compounds should be determined according to the climate conditions, region-specific host plant species, and pest species. The number of males in different traps will give you idea to establish the treatment time. Sex pheromones are useful to determine the exact management time and this will help to reduce the cost of the management strategies. The knowledge about pest and geographical distribution will improve the success of catches. Pheromone traps are useful to monitor for mostly Lepidoptera, Coleoptera, and Diptera Family. Climate changes, moth age, moth behavior, moonlight, pheromone release rate, crop cover, crop phenology effects performance of the monitoring strategy [70–72].

#### **4.2 Mass trapping**

The aim of mass trapping tactic is to eliminate males or females with semi chemicals such as host volatiles, aggregation, and sex pheromones from the target pest population to prevent population growth. Trap design, population level, biology of the pest, density, and immigration can change the success of the application. For example, regarding biology of the target pests, mass trapping is more effective when the pest is univoltine, monogamous, and monophagous instead of multivoltine, polygamous, and polyphagous species [73]. The economic viability of mass trapping as a control measure is determined by the composition of pheromones and the expenses incurred in their production. For instance, *Melanotus communis* Gyllenhal (Coleoptera: Elateridae) employs a solitary chemical, 3-tetradecanyl acetate, which influences the cost of traps and the manual labor required, ultimately affecting its competitiveness in the farming industry. In banana crops, the technique of mass trapping was employed to control two types of weevils, such as *Cosmopolitus sardidus* (Coleoptera: Curculionidae) and *Metamosius hemipterus* (Coleoptera: Curculionidae), using distinct methods. The former was captured using a pitfall trap baited with a


#### **Table 2.**

*Monitoring trials against invasive pests.*

#### *Perspective Chapter: Pheromone-Based Techniques in Sustainable Pest Management DOI: http://dx.doi.org/10.5772/intechopen.110862*

pheromone, while the latter was caught with a gallon trap baited with sugarcane and a pheromone. In plots, corn damage diminished by 61–64% during the experiment. Banana bunch weight increased 23% compared to control plots after 12 months of trapping. Trapping for *C. sordidus* over 200 hectares reduced corn damage up to 62–86%. Insecticide control measures resulted in about 20–30% corn damage while the use of pheromone trapping to manage *C. sardidus* lowered corn damage to 10% [74]. Optimization of the lures and traps [73], operational costs provide to determine the most suitable method [75, 76] but, inadequate number of traps, polygamous nature of codling moths leads to inadequate control of pests [73]. Surface lubricants [77, 78], surface conditioners (Fluon), insecticides, water, or other physical structures, increase the number of pests collected in traps [18, 78]. Lures that attract both male and female [79] by using one trap to collect different pests will improve the success of mass trapping [29].

Chinese first mass trapping test using rubber septa containing pheromones of *Grapholita molesta* was found successful. They observed that female mating rates were diminished 74% and 83% in treated orchards compared to control and fruit infestation rates per fruit reduced by 50–70% and thus reduce 50% reduction in cost of insecticide application. In China, a research group found that pheromone compounds identified for *Paranthrene tabaniformis* in the United States was not effective in the field of China. It was due to the Chinese strain of pheromones consist of compounds in different ratios when compared to United States strain [80]. The field trapping experiments were carried out by various types of trapping systems and different dosages of pheromone lures. At the end, mass trapping system resulted in a significant reduction of this pest with female mating rates decreased by 43–79% and the total pest population reduced by around 57–95%. The use of mass trapping technique was experimented on the overwintering generation of C. suppressalis on rice plants. The results showed a significant decrease of 61% in egg laying and a 57% decrease in rice plant infestation in the treated field, as compared to the field treated with pesticides. [81]. Experiments about some of the pests their host and region was given in **Table 3**.

Aggregation pheromones of predators can be used for mass trapping such as aggregation pheromones of *Coccinella septempunctata* L. (Coleoptera) can be used for the management of aphids [84]. Recently this mass trapping tactic is successful for the management of the Japanese beetle, *Popillia japonica* Newman (Coleoptera: Scarabaidae) [85].


#### **Table 3.**

*Mass trapping trials against invasive pests.*

#### **4.3 Attract and kill**

Attract and kill method used for capturing and killing the pests. Both field crop and stored product pests are controlling with this method [20]. The attractive method shares similarities with mass trapping, but its effectiveness extends to a broader area and its killing impact is not limited to a single trap [86]. When pest density and immigration rate is low, attract and kill method is working well similar to mass trapping method [73, 87, 88]. For some of the Lepidoptrean pests, attract and kill method is better when compared to mating disruption because attract and kill method does not allow males to recover or mate [89]. In the management strategy *Carpophilus* spp. aggregation pheromone was incorporated with ripening fruit and insecticide [90]. *A. grandis* that is a main pest of cotton in the USA and males produce aggregation pheromone grandlure [91]. This aggregation pheromone and control tubes provided strong reduction in pest population [66, 92]. *Musca domestica* are attracted by muscalure used with co-attractants in attract and kill approaches [93, 94]. In Switzerland 0.16% pheromone and 6% permethrin were used to control codling moth as part of the attract and kill method. Attract and kill method have been effective for *Bactrocera dorsalis*, *Bactrocera cucurbitacea* and *C. capitata* for 50 years [95]. Furthermore there are lots of studies about this attract and kill method (**Table 4**).

#### **4.4 Mating disruption**

Mating disruption prevents pests to find their mates and hinders their reproduction of them [89, 101–104]. There are four mechanisms that should take into consideration, one of them is semi chemicals leads to false trail by attracting males away from the females while the other mechanism is camouflage. The presence of semi chemicals pervades the environment, hindering the ability of males to locate females and thereby disrupting the mating process between male and female. Sensory desensitization by overexpressed semi chemicals also leads to hinder males to find females. The last mechanism leads to the emigration of males due to the excess pheromone release. Male cannot find females for mating [20, 105, 106]. Application rate, dispenser design, dispenser height, and trap density are important components for the success of the management technique. There are some important factors that should be considered due to several mating disruption dispensers and formulations. Passive dispensers are leading the way in mating disruption programs due to


#### **Table 4.**

*Attract and kill trials against invasive pests.*

#### *Perspective Chapter: Pheromone-Based Techniques in Sustainable Pest Management DOI: http://dx.doi.org/10.5772/intechopen.110862*

their ease of application and cost-effectiveness compared to other options available. But they release pheromones continuously. Time of the day and pest flight activity time should be considered. Aerosol dispensers can release pheromones at the exact time, and the amount of pheromones released per unit can be arranged. Aerosol delivery system is faster and cheaper than passive dispensers and never be affected by environmental degradation. Aerosol dispensers target multiple pest species and modern digital electronic technologies support this technique. But wind can affect the efficiency of aerosols and this technique is more effective when applied for large areas [104]. In 1953 China, *C. pomonella* pheromone lures were placed in apple orchards. After 2 years, the mating rates of female moths reduced by 55–75% and the infestation rate also decreased by 35–77%. This was the first study using mating disruption in China [107, 108].

This technique is environmentally benign, does not affect non-target organisms, and is approved for organic production system [18]. Mating disruption is a successful management strategy for *L. dispar* [109], *Cydia pomonella* [110], *Lobesia botrana* [111], *Grapholita molesta* [112], *Ephestia cautella*, *Ephestia kuehniella*, *Plodia interpunctella* [113], *Cossus insularis* [114], *Grapholita molesta*, *Keiferia lycopersicella*, *Pectinophora gossypiella*, *Platynota stultana* [11, 35, 115], *Tuta absoluta* [116], *Plodia interpunctella* (Wjayaratne), *Chilo suppressalis* [117], *Cydia fagiglandana* and *C. splendana* [118]. There are also non-lepidopteran pests *Anomala orientalis, Planococcus ficus* [119], and *Prionus californicus* that are affected by mating disruption [120, 121] (**Table 5**).

Population density is important for the success of this management technique. High density of the population is difficult to control [102, 124–127]. In one of the studies Borchert and Walgenbach (2000) mentioned that mating disruption was effective in low-density populations of the *Platynota idaeusalis* [128]. It is also reported that mating disruption application with pesticides or alone did not control *Cydia pomonella* in high-density population [126]. Attractive plant-derived kairomones with sex-releasing pheromones increased the effectiveness of mating disruption technique [129–132]. Other successful mating disruption trials in low density have been experimented for *Lymantria dispar* [35, 133, 134] and *Cydia splendana* [35, 135]. Especially landscapes such as residential areas, parks, commercial sites, with low density of pest, mating disruption technique are effective. Mating disruption technique was not found effective when males and females were close to each other in space and time [133]. The amount of pheromone lures is also important to increase moth captures. For example, it is observed that the trapping of codling moth males


#### **Table 5.**

*Mating disruption trials against invasive pests.*

increased when 10–20 mg pheromone was baited [9] or by position traps in the upper canopy [11, 35]. Exposure to a high concentration of pheromone blend lead to generating insensitive males [110] and immigration have an adverse impact on the success of mating disruption [102].

Numerous methods are utilized in mating disruption to dispense pheromones. Examples include microencapsulation, hand-applied dispensers, polymer spirals, and ropes, spray application similar to insecticides, as well as hollow fibers and twist-tie ropes. New devices which have large polymer bags loaded with large amounts of pheromones are used instead of batteries and other puffers. Pheromone release rate, different dispenser types, dispenser placement, or population density will affect the control of pests. Nevertheless, mating disruption is an excellent technique for integrated pest management programs.

In another experiment, mating disruption technique was used to control *Tuta absoluta*. To assess the efficacy of pheromone lures, six fields were monitored, with three fields treated with pheromones and three fields left as control. The number of male insects detected in traps per week was significantly lower in pheromone-treated fields compared to the control groups in both 2018 and 2019. Specifically, the mean number of males detected per week was 120 ± 16 and 69 ± 15 in the pheromonetreated fields, while it was 299 ± 16 and 230 ± 15 in the control groups, respectively. Moreover, the percentage of infestation rate was significantly lower in pheromonetreated fields than in control fields in both years. Specifically, the mean infestation rate was 4 ± 0.56% and 1 ± 0.52% in the pheromone-treated fields, while it was 11 ± 0.56% and 7 ± 0.52% in the control fields in 2018 and 2019, respectively [116].

In Southeast Germany, Ecoflex fibers have been experimented with in mating disruption trials for the management of *Lobesia botrana* [136]. Later electrospun mesofibers were became popular with their biodegradable property and also harmless to non-target organisms [137]. Nowadays, Isonet L TT was tried for 3 years in Italy and found to be successful for the control of the *L. botrana* during the whole season [138].

#### **4.5 Push-pull strategies**

Push-pull strategy is first coined by Pyke and coworkers. Repellent and stimuli were used for the management of *Helicoverpa* species in cotton [139]. Currently, this tactic is also called a "stimulo-deterrent diversion tactic." There are two types of push-pull strategies, one of them is intercropping with repulsive non-crop plants with attractive trap crop and the other one is semi chemical repellents with attractive pheromone traps. Repellents cause pests to avoid the crop (push) and attracting pests to pheromone traps that remove pests before they find mates or hosts (pull). Insect biology, chemical ecology, and interaction between host plants and natural enemies should be known to apply this strategy [140–145]. Synthetic repellents, host and non-host volatiles, host-derived semi chemicals, antifeedants, oviposition deterrents, and stimulants can be used in these strategies. Traps capture rate depends on (1) trap size and efficiency, (2) traps blends of attractive volatiles, (3) release rates, (4) population density of flying pest insects, and (5) competitive sources of natural attraction. There are some experiments about push and pull strategies for the management of the Lepidoptera family (*Helicoverpa armigera*, *S. frugiperda),* Coleoptera family (*Sitona lineatus, Leptinotarsa decemlineata, Meligethes aeneus, Dendroctonus ponderosae*), and Diptera family (*Drosophila suzukii*) which were given in **Table 6**.

*Perspective Chapter: Pheromone-Based Techniques in Sustainable Pest Management DOI: http://dx.doi.org/10.5772/intechopen.110862*


#### **Table 6.**

*Push-Pull trials against invasive pests.*

The behavior of pests is manipulated by push-pull tactics by combining stimuli for push components and stimuli for pull components. To deter pests, various methods can be employed such as altering the host's color, shape, or size. DEET (N, N-diethylmeta-toluamide) is commonly used for warding off cockroaches and lady beetles. On the other hand, volatiles like citronella and eucalyptus essential oils can be utilized to mask host odors or induce non-host avoidance. Semi chemicals, antifeedants such as azadirachtin, alarm pheromones (generally used for aphids) are also used for push components. Visual stimulants, host volatiles (Baits, HIPVs), kairomonespost volatiles, sex and aggregation pheromones, and also gustatory and oviposition stimulants can be used as stimuli for pull components. In one of the case studies to protect the maize and sorghum crops from stem borer pests. Stem borers are repelled from the crops by planting repellent nonhost intercrops like molasses grass (*Melinis minutiflora*), silverleaf desmodium (*Desmodium uncinatum*), or greenleaf desmodium (*Desmodium intortum*) which set as the "push." Then trap crops like Napier grass (*Pennisetum purpureum*) or Sudan grass (*Sorghum vulgare sudanense*)—the "pull" component are used. For the management of *Helicoverpa* in cotton, neem seed extracts are applied to the cotton crop as push strategy and pigeon pea (*Cacanus cajan*) or maize (*Zea mays*) is planted as a pull strategy. A pull strategy was employed for managing the Colorado potato beetle, wherein the beetle was attracted to host plant volatiles and a potato trap crop was sprayed with an attractant. Additionally, a push strategy was implemented by applying antifeedant neem between the rows.. Then instead of plant volatile Colorado beetle aggregation pheromone (S)-3,7-dimethyl-2-oxo-6-octene-1,3-diol was applied as pull strategy. *S. lineatus* pea leaf weevil is a pest of legumes its pheromone 4-methyl-3,5-heptanedione used as pull and commercially available neem antifeedant used as push component of the strategy. Another case study was about the management of pollen beetle *M. aeneus* which is acted on oilseed rape (*Brassica napus*). Turnip rape was planted as the pull stimuli and lavender (*Lavandula angustifolia*) was used as a push component and also alkenyl glucosinolates were used as the main attractant of such pest. Another example for a pull and push strategy is *Delia antiqua*. It is a main pest of onion. Small unmarketable bulbs were used as a trap crop as a pull strategy, cinnamaldehyde is an oviposition deterrent used as a push stimulus. So significant reduction was observed when using this push pull strategy. For the management of *Drosophila suzukii*, mass trapping was applied as a pull strategy and

1-octen-3-ol which is an oviposition deterrent was used as a push strategy. In the end high reduction in oviposition of *D. Suzukii* was observed.

There are lots of advantages of push-pull tactics over traditional pest management methods. This strategy can be used both in juveniles and adult stages of pests. Simple, commercially available, nontoxic, and cheap components can be used as a stimuli. This management technique can increase efficiency of individual push and pull components, antifeedants and oviposition deterrents are used as a push tactic and it do not need to struggle with resistance management. Besides there are some disadvantages such as limited specificity, and there are lots of odor sources for attraction. We should develop the technique by understanding the behavioral and chemical ecology of the host pest, and the development of semi chemical components. People generally choose to use insecticide applications instead of biological control agents. But this strategy is a useful tool for integrated pest management programs reducing pesticide input [151]. Enhancement of monitoring and decision-making systems is crucial, and a comprehensive approach is required to regulate the system [152].

The presence of Nepetalactone, an aphid sex pheromone constituent, and (Z)-Jasmone, a plant volatile, can have an impact on aphid parasitoids. In turn, these parasitoids can offer a natural means of controlling aphid populations. While tricosane and pentacosane lady beetle pheromones provide to push the parasitoids from surrounding areas to the treatment crop. These two lady beetle pheromone components (tricosane and pentacosane) used by the *Aphidius ervi* which is an aphid parasitoid to escape from seven spotted lady beetle [153].

Another example for push-pull strategy is about bark beetles (Scolytidae). Antiaggregation pheromone 3-methylcyclohex-2-en-1-one and aggregation pheromone (frontalin, seudenol, 1-methylcyclohex-2-enol and ethanol) diminished populations of the *D. pseudotsugae* [154]. Push-pull strategy is also applied with aggregation and anti-aggregation pheromones for the management of other forest pests that are *D. ponderosae, Ips paraconfusus*, *Dendroctonus frontalis* [142]. "Verbenone" is an anti-aggregation pheromone that reduces the attract rates of *D. ponderosae* Hopkins (Coleoptera: Curculionidae) [155–159]. Trees baited with aggregation pheromone grandisoic acid and fruit volatile benzaldehyde attracted plum curculio adults. It is also mentioned that instead of stand art insecticide application pheromone-baited "trap" trees provided satisfactory suppression of fruit injury [37]. As a result, 93% fewer trees were sprayed with insecticide with this "trap" tree approach [37].

Chemical ecology, geographical variation can change aggregation pheromone components [160]. For instance Australian and the Hawaiian populations of the *Rhabdoscelus obscurus* Boisduval produce male specific 2-methyl-4-octanol. This compound can enhance the attractiveness of Hawaiian populations only. Australian population (E2)-6-methyl-2-hepten-4-ol (rhynchophorol) combined with 2-methyl-4-octanol to attract the Australian males. This experiment shows that interactions between organisms and their pheromone according to their regions should be studied.

#### **5. Conclusion**

The intensive use of pesticides leads to residue problems on food, resistance of pests, environmental pollution, and health problems. Pheromones can be used safely instead of broad-spectrum insecticides in sustainable management strategies. Pheromones are volatile and do not leave residue behind, species-specific and cost-effective and do not cause toxicity. By using pheromones, male adult number,

#### *Perspective Chapter: Pheromone-Based Techniques in Sustainable Pest Management DOI: http://dx.doi.org/10.5772/intechopen.110862*

mating rates can be reduced and also insecticide application time can be detected by monitoring strategy. Nowadays studies are mainly about identification, optimization of pheromone components of region-specific species, and also development of new formulations. Active compounds extracted from insect frass can be used as repellent or deterrent for the conspecifics. Besides new strategy is about odorant receptor genes which allows discovery of compounds that can trigger or block these receptors. Plant-insect interactions also should be studied and enzymes involved in the odorants and pheromones degradation should be observed to interfere the chemical communication of insect. The ultimate change will be to improve the process by employing simulation models. One factor of concern is high cost of the pheromones in comparison to pesticide application. For this reason producers, farmers and scientists should reduce the cost of application of pheromones to affordable threshold. Hence they should target low population density, to release pheromone in exact time of flight, to arrange the pheromone release rate, to develop more efficient formulations regarding the ecological conditions and also they should target to use multiple pheromones to monitor several pest simultaneously. Successful implementation of pheromone based management techniques can diminish economic cost and lighten the ecological footprints as this management technique decreases insecticide usage.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Melis Yalçın Faculty of Agriculture Engineering, Adnan Menderes University, Aydın, Turkey

\*Address all correspondence to: melisusluy@adu.edu.tr

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[158] Gilette NE, Mehmet CJ, Mori SR, Webster JN, Wood DL, Erbilgin N, et al. The push-pull tactic for mitigation of mountain pine beetle (Coleoptera: Curculionidae) damage in lodgepole and whitebark pines. Environmental Entomology. 2012;**41**:1575-1586. DOI: 10.1603/EN11315

[159] Progar RA. Five year operational trial of verbenone to deter mountain pine beetle (*Dendroctonus ponderosae*.i Coleoptera:Scolytidae) attack of lodgepole pine (*Pinus contorta*). Environmental Entomology. 2005;**34**:1402-1407. DOI: 10.1603/0046-225X-34.6.1402

[160] Giblin-Davis RM, Gries R, Crespi B, Robertson LN, Hara AH, Gries G, et al. Aggregation pheromones of two geographical isolates of the New Guinea sugarcane weevil, *Rhabdoscelus obscurus*. Journal of Chemical Ecology. 2000;**26**:2763-2780. DOI: 10.1023/A:1026

### **Chapter 4**

Perspective Chapter: Tapping Significance of Microbial Surfactants as a Biopesticide and Synthetic Pesticide Remediator – An Ecofriendly Approach for Maintaining the Environmental Sustainability

*Shikha Gaikwad*

#### **Abstract**

Pests are the major concern for plant infections that affect the agriculture production drastically and result in the loss of economy. Regular use of synthetic chemicals develops resistance in pests and affects soil, plant, and human health. The development and promotion of green technology facilitated by microbiota helps in maintaining a healthy environment. Molecules of microbial origin are well-known elicitors for stimulating and sustaining the plant immune system and fertility level of the soil. They compete with the pathogens for resources like food and space, activate the inherent defenses of plants, synthesize antimicrobial chemicals, or other metabolites that degrade and remediate synthetic chemicals. Biosurfactant (BS) is an important amphiphilic molecule with polar and non-polar. Its structure contributes to its high biodegradability, low toxicity, and stability in harsh environments. In the chapter, the multifunctional properties of biosurfactants, methods used for evaluating their biosurfactant producing abilities, methods used for identification, and characterization of the chemical structure of biosurfactants, along with the significance of metagenomics documented. The mechanisms of biosurfactants in controlling the growth of pests and their importance as pesticide remediations are also discussed.

**Keywords:** biosurfactants, biocontrol agent, biopesticide, pesticide remediator, metagenomics techniques

#### **1. Introduction**

All countries have concern about meeting the expanding food needs, which places an additional burden on the agricultural and food business. Controlling the spread of

phytopathogens and raising the soil's fertility level have been top priorities in efforts to increase crop output. To cater to ever-increasing demand, chemical fertilizers, insecticides, and fungicides are continuously used for decades. Pesticides at the rate of 2 million tons are used annually, with 2% being rodenticides and nematicides and 50% being herbicides, insecticides, and fungicides [1]. These pesticide residues endure in the water and soil for years and develop pesticide resistance among deleterious microbes [2–4]. It has been reported that milk, meat, and other food products contain considerable quantities of pesticide residues [5]. Boedeker with his coworkers highlighted, harmful effects of synthetic chemicals on almost 44% of the farming population every year [6]. The most prevalent morbidities that are connected to these practices include immune system deficits, pulmonary dysfunction, and malignancy [7, 8]. The primary cause of these consequences may be the overuse of agrochemicals, which has been a considerable issue [9]. To enhance soil fertility and prevent insect infestation, it is necessary to consider the revitalization of native soil systems that will resist the use of these synthetic chemical amendments. Finding solutions that hold sustainability in the environment is required urgently. Numerous physical, chemical, and biological strategies are frequently used to solubilize and/or degrade hazardous substances. The use of membrane filtration, adsorption, soil washing, granular activated carbon, and photocatalytic remediation are some of the common examples of physical approaches. Ion-exchange, precipitation, coagulation, floatation, and flocculation procedures are examples of chemical-based methods. These physicochemical techniques that are in use at frequent intervals are relatively futile and unsustainable. Whereas combinations of physicochemical techniques occasionally prosper [10]. It is reported that synthetic surfactants are combined in many pesticide formulations as adjuvants [11]. Synthetic surfactants are expensive, caustic, and impervious to degradation. Hence a green approach that conforms to the strict guidelines of green chemistry and green technology, which are in high demand in the modern period, is essential. It will support to maintain and sustain the desired healthy flora and fauna, which can deliberately keep the ecosystem healthy.

#### **2. Overview of biosurfactants**

Surface-active substances known as biosurfactants (BS) are synthesized by some of the prokaryotic and eukaryotic microorganisms in their stationary stage of development. According to a recent survey by Global Market Insight, between 2020 and 2026 year, the BS market is anticipated to expand at a rate of more than 5.5% Compound Annual Growth Rate. In 2019, the market for BS was worth more than USD 1.5 billion [12]. Microorganisms generate BS either extracellularly or as an ingredient of the cell membrane [13, 14]. They are prevalent because of their wide range of benefits against synthetic surfactants (**Figure 1**).

BS nature is highly dependent on the microbial origin and on the available nutrients. In accordance with their chemical complexity or molecular weight, they are categorized as high and low molecular weight BS. Their molecular weights range from 50 to 1500 kDa LWM-BS (Low Molecular Weight Biosurfactants) decrease the surface and interfacial tensions at the air and water boundaries. In disparity, HMW-BS (High Molecular Weight Biosurfactants) also described as "bioemulsans," are efficient at stabilizing the oil in water emulsions (**Table 1**).

Based on the chemical structure, it is an amphiphilic molecule, with a polar head and nonpolar tail. The hydrophilic moiety is made up of intrinsically simple ester, phosphate, hydroxyl, or carboxyl groups, as well as carbohydrates like

*Perspective Chapter: Tapping Significance of Microbial Surfactants as a Biopesticide… DOI: http://dx.doi.org/10.5772/intechopen.111747*



#### **Figure 1.**

*Biosurfactants strengths and weakness of chemical surfactants.*


#### **Table 1.**

*Differentiating points of biosurfactants and bioemulsifiers.*

monosaccharides, oligosaccharides, and polysaccharides, as well as proteins, amino acids, and peptides. Unsaturated or saturated fatty acids, hydroxyl fatty acids, or fatty alcohols make up the hydrophobic portion (**Figure 2**).

Based on its structural characteristics, BS has several advantageous properties which enable them to be employed as an adjuvant or essential component in a wide range of formulations, as a potential bioremediator for synthetic chemicals, and as an emulsifier in an eclectic assortment of industrial applications, such as those involving food and beverage, petroleum, cosmetics, organic chemicals, and

#### **Figure 2.**

*(A) An amphipathic structure of a biosurfactant, (B) surfactant monomer at the surface of water and (C) micelle formation above CMC (critical micelle concentration).*

pharmaceuticals [15]. BS occupies a unique place in the agricultural sector due to its capacity of emulsification, dispersion, solubilization, foaming, and wetting agents, which accelerate hydrophobic molecules' solvation in aqueous media for the formation of emulsion [16–18]. They have proved themselves to be a top contender for leading the way in the field of agricultural and environmental science [19–28].

### **3. Multifunctional properties of biosurfactants**

BSs' amphipathic structure is giving them a special place to showcase their multifunctional properties [16]. A few important multidimensional properties are mentioned in **Figure 3** and detailed description is listed below:

#### **3.1 Surface and interfacial activity**

Surface tension is a phenomenon that happens when a liquid surface and another phase come together (it can be a liquid as well). The molecules at a liquid's surface are drawn toward the liquid's center by the contracting force of surface tension. The surface and interfacial tensions of certain fluids can be reduced by BS at extremely low concentrations due to lower Critical Micelle Concentrations (CMC).

**Figure 3.** *Functional properties of biosurfactants.*

*Perspective Chapter: Tapping Significance of Microbial Surfactants as a Biopesticide… DOI: http://dx.doi.org/10.5772/intechopen.111747*

#### **3.2 Solubilization**

In the aqueous phase, biosurfactant molecules spontaneously group together and form supramolecular nano sized aggregates with a hydrophobic core and a hydrophilic surface exposed to water. It is formed when BS concentration surpasses a threshold value which is well known as critical micelle concentration—CMC. These small sized aggregations enhance the bioavailability of water-insoluble substances in aqueous liquids or chemical agents by allowing the transportation and confinement of insoluble molecules.

#### **3.3 Wetting**

It is the capability of a liquid to provide a link with another surface and to spread out evenly and easily over the top of the surface of another solid or fluid. When a liquid with a high wetting capacity spread across a surface, it creates a thin, continuous film. Biosurfactants are effective wetting agents because they lower liquid surface tension by reducing attractive forces, which enhances affinity toward different surfaces. Instead of connecting them to the surface tension, it penetrates the pores.

#### **3.4 Foaming**

Small levels of BS can decrease a liquid's surface tension (lessen the effort required to produce foam) or boost its colloidal stability by preventing bubble coalescence. Biosurfactants are intense at the gas-liquid interface, where they form fizzes that move through the liquid and produce foam. BS is a substance that encourages the production of foam.

#### **3.5 Dispersion**

Dispersion results from the decrease in the cohesive attraction between similar particles. A few BS are applied as a dispersion agent to reduce the aggregation of insoluble particles amid each other in the suspension. For example, BS removes hydrophobic molecules from the surfaces of rocks to improve their mobility and recovery in oil extraction steps. Dispersion also plays an additional role to reduce or completely eradicate the biofilm formation of undesirable microbes.

#### **3.6 Flocculation**

The process of flocculation starts when colloidal particles, either natively or because of the addition of a clarifying agent, migrate from a condition of suspension to sediment in the form of floc or flake. Emulsions are restored to their original state by these flocs, which can be distorted by mechanical force and aren't permanent.

#### **3.7 Emulsification**

It is the process of combining liquids that ordinarily do not mix to create an emulsion. BS has emulsifying and demulsifying properties. The two most prevalent emulsions are water-in-oil (w-0) and oil-in-water (o-w). They are typically unstable in two-phase solutions. Biosurfactants signifies the solubilization of large particles with micellar structures by assisting the dispersion of one liquid into another and making it easier for two immiscible liquids to be mixed.

#### **3.8 De-emulsification**

In this process, the stable interface between the internal and bulk levels gets disturbed, due to which the emulsions get split. BS makes the process of de-emulsification easier.

#### **3.9 Biodegradability**

BS is a metabolic byproduct of microorganisms and is easily broken down in nature without generating any deleterious byproducts.

#### **3.10 Biocompatibility, low toxicity, and digestibility**

They are not harmful or toxic to living tissue. When they are interacting with other organisms, they do not change their bioactivity or mechanism.

#### **3.11 Stability at various extreme conditions**

Several BS stay stable across a diversity of adverse environmental situations, such as pH, temperature, and salinity, rendering them potential candidates to be deployed in a comprehensive array of industries.

#### **4. Methods to detect biosurfactants producing ability of a microbial strain**

To choose biosurfactant synthesizers from a set of microorganisms, various screening techniques are advocated concurrently (**Figure 4**). A single evaluation system is never recommended [29], various techniques that can be used are:

#### **4.1 Surface tension and interfacial measurement**

The aspects of surfaces and interfaces, such as the surface excess concentration, adsorption kinetics, surface pressure, and CMC can be recognized with the aid of tensiometers. These instruments differ in terms of the underlying physical concepts, the mechanical layout, the type of measurements they can perform—static or dynamic and whether they can detect surface tension (ST), interfacial tension (IM), or both. It is possible to follow the adsorption kinetics by making dynamic measurements on surfaces or interfaces that are not in equilibrium. The most common techniques that are used to evaluate ST, IM or both are Du Noüy ring, Wilhelmy plate, and pendant drop. The strength required to separate a wire ring or loop from a surface or interface is the cornerstone for these strategies. The detachment force and interfacial tension have an inverse relationship. The accuracy, simplicity, and minimal sample volume are needed to make this approach more advantageous. It is the easiest screening technique and is suitable for a preliminary screening.

#### **4.2 Drop collapse method**

Polar water molecules are attracted away from the hydrophobic surface in the lack of surfactants, which helps to stabilize the drops. In comparison, when there are surfactants in the liquid, the drops spread or even collapse due to the reduced force

*Perspective Chapter: Tapping Significance of Microbial Surfactants as a Biopesticide… DOI: http://dx.doi.org/10.5772/intechopen.111747*

#### **Figure 4.**

*Methods to detect biosurfactants producing ability of microbes.*

or interfacial tension. Drop stability is correlated with surface and interfacial tension and is reliant on surfactant content. It is easy, quick, and simple, and needs a little volume of sample and no specific equipment.

#### **4.3 Penetration assay**

This test requires contact between two insoluble phases to function. A staining solution is applied to culture supernatants for easy differentiation. If BS is present, the colored supernatant passes through the oil film barrier and enters the paste. Within 15 min, it transforms the color to murky white. Supernatant devoid of biosurfactants would lose its color when becoming cloudy. It is a quick and effective method to screen through many potential isolates.

#### **4.4 Agar plate overlaid with hydrocarbons**

In an oil coated agar plate, an axenic culture is streaked and incubated for 5–7 days at a desired temperature. An emulsified halo around the streaked colonies can be identified as BS producers.

#### **4.5 Aximetric drop shape analysis (ADSA)**

The technique includes assessing the drop's shape, which is strongly impacted by the equilibrium between surface tension and outside forces, like gravity. The shape of the drop exemplifies its range of utility and validity.

#### **4.6 Cell surface hydrophobicity technique**

It is based on a technique that employs microbes to attach to hydrocarbons. Microorganisms that can actively absorb hydrocarbons typically have highly hydrophobic coverings.

#### **4.7 CTAB (cetyltrimethylammonium bromide) agar plate method**

It is used basically to identify their anionic, cationic, nonionic, and neutral nature. The media is supplemented with different dyes such as cetyltrimethylammonium bromide and methylene blue. Presence and absence of a halo zone surrounding the culture reflects its nature and property of the BS.

#### **4.8 Hemolytic activity**

In media such as Luria agar (LA) or nutrient agar (NA) fresh blood is mixed. Microbe is streaked and incubated at the desired temperature. After incubation hemolysis zone was observed around the colony which indicates BS synthesizing ability of microbes.

#### **4.9 Emulsification index (EI)**

Emulsification activity is calculated by EI. Hydrocarbon is added to the culture broth and mixed. Subsequently allowed to stand overnight. The height of the emulsion formed between aqueous, and hydrocarbon is measured, that defines the stability and strength of a surfactant.

#### **5. Methods to identify and characterize biosurfactants**

New approaches are evolving for recognizing and identifying BS because of scientific and technological innovations. The techniques that are used to identify and characterize the BS are as follows (**Figure 5**).

#### **5.1 Thin layer chromatography (TLC)**

The most popular, straightforward, and affordable approach for identifying various macromolecules, including carbohydrates, lipids, fats, proteins, amino acids, and peptides. In this methodology, an adsorbent medium such as aluminum oxide (alumina), silica gel, or cellulose is wrapped thinly around a sheet of glass, plastic, or glass. The sample is placed on a plate and given time to go through several mobility phases. The development of the colors after spraying developers helps in recognizing the presence of macromolecules that help in identifying the class and subclass of BS.

#### **5.2 High performance-liquid chromatography (HPLC)**

It is the most comprehensive and efficient quantitative tactic. A detector, a mobile phase, and a fixed phase constitute it. The mobile phase moves the sample solution once it is introduced through the injector port. The mobile phase progressively lowers the components of the sample solution over the stationary phase, which is a solid. The migration of the components is regulated by the noncovalent interactions between the compound and the column. This methodology considers several components according to their polarity. The segregated products may then be recognized, and fractions for certain peaks can be obtained to investigate the structure of BS moiety.

*Perspective Chapter: Tapping Significance of Microbial Surfactants as a Biopesticide… DOI: http://dx.doi.org/10.5772/intechopen.111747*

#### **Figure 5.**

*Methods to identify and characterize biosurfactants.*

#### **5.3 Gas chromatography-mass spectrometry (GC-MS)**

Mass spectrometry, coupled with either gas or liquid chromatography, is utilized to discover the chemical bonds and structure of BS. In addition, the procedure provides a qualitative and quantitative approach used for detecting the chemical nature of BS. The mass spectrometers are made up of three important components: an ion source, a molecular mass analyzer, and a detector. As a result, volatile samples are directly introduced into the device, whereas non-volatile samples need to be dissolved in volatile solvents. As the sample passes through the electromagnetic field, it immediately becomes ionized. The computer system receives the amplified automated signal and reports it as various chromatogram peaks. It is therefore essential to evaluate the overall quantity and quality of the compounds. The BS compound's hydrophobic component (water-repelling) is typically revealed by GC-MS.

#### **5.4 Liquid chromatography coupled with electro-spray ionization tandem mass spectrometry (LCESI-MS/MS)**

The structural makeup of the hydrophilic moiety is revealed by LC-MS (water-loving). Different research centers are currently using these approaches to detect BS biomolecules, which reduces the chance of erroneous characterization. It is defined as a less expensive and less time-consuming procedure and detects BS even at low concentrations.

#### **5.5 Fourier transform infra-red (IR) spectroscopy**

It is the most typical type of infrared spectroscopy. According to the underlying hypothesis, some infrared (IR) light is absorbed when it travels through a sample. Molecules with covalent bonds will selectively absorb different wavelengths of light, affecting the bond's vibrational energy. The type of vibration (stretching or bending) depends on the atoms in the linkage. The quantity of radiation that hits the sample is calculated. The spectrum is represented by plotting transmittance and wavenumber (cm<sup>−</sup><sup>1</sup> ). It is utilized in the range of around 4000 and 400 cm<sup>−</sup><sup>1</sup> . It detects and

discriminates the spectra between molecules. The sample is not destroyed, and it is substantially faster, more sensitive with an inexpensive strategy.

#### **5.6 Matrix-assisted laser desorption/ionization-time of flight mass spectroscopy (MALDI-TOF)**

This method is based on a laser energy-absorbing matrix that creates ions from large molecules with little fragmentation. The MALDI approach is a three-step procedure. The sample is first applied to a metal plate after being blended with a suitable matrix material. After that, the sample is exposed to a pulsed laser, which detects the sample and matrix material. The analyte molecules are then accelerated into a certain mass spectrometer that is being utilized to examine them. After detection, molecules are finally ionized by protonation or deprotonation in the hot plume of gases. Although the process is robust, its reliability is offering its special place in the analytical world. It has been used to analyze molecules like DNA, proteins, peptides, carbohydrates, polymers, and dendrimers that have a propensity to be brittle and fragment when ionized by more conventional ionization techniques.

#### **5.7 Nuclear magnetic resonance (NMR)-spectrometry**

It is constructed on transformations that emerge in magnetically significant atoms when an outside magnetic field exists. In a process nucleus absorbs radio frequency radiation, due to which the nuclear spin realigns or splits in the higher-energy direction and emits radiation again and returns the molecule again to the lower-energy state. For each nucleus, the magnetogyric ratio serves as a proportionality factor. NMR can be used to identify the functional groups and linkages within lipid and carbohydrate molecules. The specific location of each functional group and facts about the structural isomers can be revealed by a series of NMR spectroscopy.

#### **5.8 Fast atom bombardment-mass spectroscopy (FAB-MS)**

It relies on ionization from the liquid phase, with the probable requirement that the sample molecules congregate at the surface of the liquid matrix in the vacuum. It highlights the BS's lipid structure.

#### **5.9 Metagenomics to identify biosurfactants producing genes**

"Metagenomics" is a combination of "meta" and "genomics," which mean "the study of the microbial genome,". In it, microbiota from different environmental samples is identified, and characterized. This technique helps us to explore the sequences of the culturable and unculturable microbial community that can be explored for the betterment [30–32]. The primary processes in metagenomics involve extracting the entire genome, fragmenting the collected DNA with restriction enzymes, and putting it into an appropriate expression vector. It has been established that the gene codes for the proteins and/or enzymes participating in the biosurfactant synthesis pathway [33] are generally aggregated in the region of the chromosome, and the gene cluster is conjured up of between 3000 and 7000 base pairs. There are metagenomic libraries available for biosurfactants, including function-based techniques like Substrate-Induced Gene Expression-(SIGEX) and High-Throughput-(HTP) screening [34–36]. It is reported that the genomic research is integrated with bioinformatics such as phylogenetic

#### *Perspective Chapter: Tapping Significance of Microbial Surfactants as a Biopesticide… DOI: http://dx.doi.org/10.5772/intechopen.111747*

analysis, taxonomic profiling, molecular phylogeny, functional characterization of metagenomes, enzyme research, and system biology studies, including genetic engineering utilizing CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or Transcription Activator-like Effector Nucleases (TALENs) [37] that enhances structural and functional diversity knowledge about BS. There is a system database known as "BioSurfDB", that has compiled 58 pathways, 96 biosurfactants, 1077 microorganisms, 3736 genes, and 3430 proteins [38]. Genetic engineering techniques, however, only create small or single-gene alterations. To synthesize a good quantity of biosurfactants, more experiments that are based on optimizations are required. New regulatory aspects should be explored, and biosurfactant-producing genes should be transferred to indigenous microorganisms by exploring the importance of CRISPR-based methods [31], that are residing in various habitats which need more attention in the present era.

#### **6. Biosurfactants as biopesticide and their mechanisms**

Pests including insects, flies, rats, nematodes, bacterial and fungal infections, and unfavorable plants influence the health, cultivation, and crop yields. Surfactants have considerable structural features because they are employed as adjuvants in the preparation of pesticides to strengthen their efficacy [27]. They are widely used to prevent the growth of the fungus *Aspergillus* species, which causes aflatoxin contamination of crops [39] and to exterminate weed species that reduce land production [40]. On the other hand, its frequent use affects the health of the soil, human, and environment in a drastic manner. The reason for such a cause is its perilous, persistent, recalcitrant, and extended half-life properties. As a result, it is a challenge for researchers and scientists to find an eco-friendly approach to control this harm and pressure on the environment. Green chemistry, which may create highly valuable products and advance green technology, can overcome such problems. By suppressing the proliferation of pests or solubilizing insoluble compounds, the secondary metabolites generated by microorganisms are well recognized for their beneficial aspects on soil and plant health [25, 41, 42]. However, obtaining and using these surfactants from microbes can be a sustainable strategy for maintaining plant, soil, environmental, and human health. There are several biosurfactants derived from microbes that show antimicrobial activity against deleterious microbes that affect plants. As a result, they are proving themselves as promising biocontrol molecules. The various mechanism that highlights the efficiency of BS to be use as biopesticide are as follows:

#### **6.1 Impact on phytopathogens**

Antagonistic effects of BS are seen on various plant pathogens such as *Trichophyton rubrum, Colletotrichum gloeosporioides*, *Corynespora cassiicola, Fusarium verticilliodes, Fusarium oxysporum* f. sp. pisi, and [43], *Aspergillus flavus*, *Penicillium roqueforti*, and *Colletotrichum demiatium* [44, 45], *Fusarium graminearum*, *Botrytis cinerea*, and *Podosphaera fusca* [46–48]. *Colletotrichum capsici* [49], *Macrophomina phaseolina* [50], *Fusarium verticillioides*, *Penicillium* sp., and *Aspergillus* sp. [51].

BS inhibits the growth of these pathogens by formation of an antibiofilm layer on various surfaces, which lowers their capacity to connect, structural failure to the intercellular net and conidiophores, and deferred or lacking sporulation, which leads to a reduction in biomass production [52]. BS also causes cellular membrane destabilization, that disrupts the feeding cycle, and eventually, cell rupture leading to lysis [53, 54]. Biosurfactants'

fatty acid components modulate the protein and lipid composition of phytopathogens' cell membranes, altering the osmolarity of the cell and its cell wall structure, and making the pathogens highly receptive to them [55–57]. According to Toral [58] and Hansen [59], BS causes morphological changes in the fungal structure known as hyphae, including swelling of the hyphae, altered mitochondrial organization, lower intracellular pH, esterase activity, and decreased hydrophobicity. BS exhibits more activity against spores [51] than it does against mycelia; this discrepancy in activity may be caused by the differing compositions of mycelia and spores' cell walls [60]. An alternate method to lessen infestations with severe phytopathogens is to strengthen plants' innate immunity that can be seen by BS implementation [61].

#### **6.2 Impact of insects**

It represses the short-term attachment of hydroid larvae (*Dynarnena pumila, Obelia loveni* and *Drosophila melanogaster*) and counteracts an attachment and contractility [62–65]. In aphids it damages their cuticle membranes.

#### **7. Biosurfactants and their mechanisms involved in remediating synthetic pesticides**

During green revolution, farmers used chemicals such as fertilizers and pesticides (insecticides, herbicides, rodenticides, and fungicides) to satisfy the demand for food. It was noticed only 1% of sprayed pesticides kill their intended target species; the remaining 99 interacts with the soil and produce more complex metabolites that damage the ecosystem and notably the soil quality and human health [66]. Pesticides strongly adsorb organic matter from the soil, preventing it from desorbing. Most pesticides are nonpolar chemicals with hydrophobic characteristics that make them water insoluble [67]. The recalcitrant nature of pesticides contributes to a variety of central nervous system problems. In addition to this, pesticides have a huge mutagenic and carcinogenic potential which can result in conditions affecting fertility, skin, and eye problems. It is documented, every year 355,000 persons die from accidental poisonings, which are linked to excessive exposure and inappropriate exposure to dangerous substances [68].

The biodegradation of pesticides by microorganisms through the production of their metabolites has been investigated for the past two decades. Macro and microorganisms use it as it is readily available in ecosystems [18, 69] where many times they consume it as a source of food. When these microbes utilize it, they produce significant metabolites. One characteristic that distinguishes biosurfactants is hydrophilic-lipophilic balance (HLB), which regulates the hydrophilic and hydrophobic constituents balance in surface-active substances [70, 71]. BS is one such prominent metabolite, when present at amounts above CMC, biosurfactant-produced micelles may enhance the solubility and bioavailability of hydrophobic pesticide compounds by lowering interfacial tension and surface tension [72, 73]. Biosurfactant activities rely on the concentration of surface-active molecules until the critical micelle concentration (CMC) is attained. To lower surface tension, efficient biosurfactants have a low CMC and necessitate less biosurfactants [70, 74]. Desorption from soil particles lowers surface tension, facilitating the process of deterioration [75]. One of the most prevalent mechanisms that contribute greatly toward the bioremediation of pesticides by biosurfactants contains counter-ion binding, electrostatic interactions, ion exchange, and precipitation-dissolution [76, 77]. As a result, the soil is made productive, pollution*Perspective Chapter: Tapping Significance of Microbial Surfactants as a Biopesticide… DOI: http://dx.doi.org/10.5772/intechopen.111747*

free, and fit for agricultural cultivation [78, 79]. A natural, budget-friendly, and environmentally sustainable method of degrading pesticides and other xenobiotics on-site is only possible through microbial biosurfactant-based remediation [18]. Although most research highlights where a single bacterium is used in bioremediation (particularly culturable ones), while only a few reports highlight the use of microbial consortia [72, 80]. The capabilities of biosurfactants to remediate environmental pollutants consequently, the burgeoning environmental safety campaign toward greener technologies are the only concerns of the times [25, 26, 78, 81, 82] that can help goal of European Commission to reduce 50% pesticide pollution by 2030.

#### **8. Conclusion and prospects**

To assure the prerequisite of food for a growing population is becoming a big challenge at a global level. Regular use of synthetic chemicals is not always recommendable as they have detrimental impacts on the health of plants, soil, and humans. New strategies to enhance agriculture and food production have appeared in the last decades. Microbes and their metabolites always occupy a special place in all related domains of Biotechnology and allied Sciences. Their stability and diversity always accomplish the goals of sustainability. They also play important role in change in climate action. Among all metabolites reported till date, the multifunctional properties of biosurfactants are making this molecule popular in various domains. Therefore, it is advocated that a cutting-edge notion for evaluating a new, safer, and healthier agricultural and environmental model should be explored more and more.

In the chapter a comprehensive information about various methods employed to identify the biosurfactant ability of the microbe, methods to characterize the chemical structure is discussed. The various mechanisms such as competition, parasitism, antibiosis, induced systemic resistance, and hypovirulence by which BS controls the growth of pests are mentioned. Importance of BS as adjuvants in pesticide formations and their mechanisms in pesticide remediation is described. It is recommended that more attention should be given to this molecule and its mechanisms can be explored for other harmful pests which are not documented to date. Its high stability to varied environmental conditions can be used year-round which highlights its additional advantage in the era where a change in climate is also an immediate concern.

#### **Acknowledgements**

SG would like to thank Dr. Vishwanath Karad MIT-World Peace University.

#### **Conflict of interest**

The author declares no conflict of interest. The author has contributed to the chapter and agreed for its publication.

*Insecticides – Advances in Insect Control and Sustainable Pest Management*

#### **Author details**

Shikha Gaikwad School of Biosciences and Technology, Dr. Vishwanath Karad MIT World Peace University, Pune, Maharashtra, India

\*Address all correspondence to: drshikhagaikwad@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Tapping Significance of Microbial Surfactants as a Biopesticide… DOI: http://dx.doi.org/10.5772/intechopen.111747*

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#### **Chapter 5**

## Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects

*Rabbiah Manzoor Malik, Sahar Fazal, Narjis Khatoon, Muneeba Ishtiaq, Saima Batool and Syed Tauqeer Abbas*

#### **Abstract**

Insecticide resistance is an inherited change in pest population exposure to a specific insecticide or group of insecticides. Overuse, misuse, and high interbreeding rates have led to insecticide resistance. Genomic technologies reveal mechanisms of resistance, including decreased target-site sensitivity and increased detoxification. Genomic projects have cloned and identified targeted genes in Drosophila melanogaster and studied resistance-associated mutations in various pest insects. Advancements in genome sequencing and annotation techniques have explored complex multigene enzyme systems, such as glutathione-S-transferases, esterases, and cytochrome P450, which facilitate insecticide resistance. Identifying specific genes involved in resistance and targeted genes is essential for developing new insecticides and strategies to control pests. Insects with resistance metabolize insecticidal compounds faster due to increased catalytic rate and gene amplification. So, system biology plays a very important role in the insect resistance against insecticides and different chemicals such as DDT and permethrin. From system biology, not only the identification of genes was done, but also the protein-protein interactions were found out, which were responsible in the insect resistance.

**Keywords:** insecticides resistance, system biology of insecticide resistance, p450 and insecticides resistance, genomics of insecticides, proteomics of insecticides

#### **1. Introduction**

The most diverse group of animals on Earth is insect, which performs a lot of significant roles. The important functions of the insects are that they act as decomposers of the dead organisms, they are the necessary components of the ecosystems, they are helpful in the pollination of the plants, they also spread the seeds, they are a good source of proteins for the livestock, and they provide us with a variety of products such as dye, silk, wax, and honey [1–4]. Though insects are playing an important role in the lives of humans and livestock, they also prove to be very harmful for the environment and humans [5]. The negative effects of insects on the environment and humans are the major threats for the ecosystem. The most highlighted negative

impact of insects is on agriculture. Some insects such as locusts, caterpillars, and grasshoppers act as pests for crops because they eat the fruits, seed, and leaves of the crops. Some insects affect the development and the growth of the crops and make the plants vulnerable to the diseases; these insects include thrips, weevils, and aphids. Some insects such as locusts damage the crops, which results in famine situations. The pests have negative impacts on the agricultural crops as they spread and carry different diseases of the plants [5, 6]. These harmful impacts of the insects on the crops are resulting in the food shortage in the different parts of the world because the population of the world is increasing rapidly. The damages caused by the insects are also increasing with the increase in climate change [7]. Insects are the main source of causing the infectious diseases among the humans and livestock; for example, mosquitoes are responsible for causing malaria and dengue. Other insects involved in causing infectious diseases are kissing bugs, head lice, body lice, tsetse flies, and so on [8]. Keeping in view these all negative impacts of insects on the humans and environment, there is a need to control the insects.

#### **2. Control of insects and insecticide resistance**

Insecticides are used for the control of insects (such as termites, cockroaches, lice, and mosquitoes) in public health, industries, households, and agriculture. Firstly, the DDT was used to control the insects, but the insects got resistant to DDT, and it was reported in the houseflies in 1947. The new and most widely used insecticides are carbamates, organophosphates, formamidines, neonicotinoids, and pyrethroids. These insecticides were very effective against the insects in the beginning, but with the passage of time, insects became resistant to these insecticides [9, 10]. An inherited change in the exposure of a population of a pest to a certain insecticide or a group of insecticides is called insecticide resistance. The insects that cannot be controlled by the repeated use of a particular insecticide are said to be resistant to that insecticide [11]. The insects are getting resistant to the insecticides quickly because the insecticides are overused or misused, the insects' population is greater in size, and they interbreed at a very high rate [12].

#### **3. Genomics and its significance in the field of biological sciences**

An interdisciplinary field of biology that focuses on the function, structure, mapping, genome editing, and evolution is called genomics. The complete set of the DNA of an organism is called the genome. The aim of genomics is to study all the collective quantification and characterization of the genes of an organism and their impact on an organism [13]. The field of the biological sciences has become advanced with the help of the genomics as it involves the analysis and the sequencing of the genomes by using the next-generation sequencing and computational tools to analyze and assemble the structure and functions of the genomes [14]. Genomics has brought a revolution in the field of biological sciences such as systems biology, discovery-based research, biotechnology, medical diagnosis, personalized medicines, identifying therapeutic targets, forensics, biology systematics, and finding the evolutionary histories of the organisms. Intra-genomic studies are also involved in genomics such as pleiotropy, epistasis, heterosis, and the interactions between the alleles and loci within the genome [15].

*Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects DOI: http://dx.doi.org/10.5772/intechopen.112662*

#### **4. Genomics and insecticide resistance**

The technologies of genomics are showing different mechanisms of insecticide resistance, which involves decreased target-site sensitivity and increased detoxification [16]. Some possibly important concerns related to the quick insecticide resistance among insects with the evolutionary time are also revealed by the genome projects. Evolutionary biologists are being provided with contemporary and ideal model systems to study the evolution of the resistance among insects for the insecticides [17, 18]. The use of the tools of molecular biology to eliminate the mechanisms of insecticide resistance is of great interest. In 1990s, traditional techniques of molecular biology were used to investigate a few cases of insecticide resistance at a molecular level. The cases that involved the known genes could easily be cloned with the heterologous PCR were manageable. From the early studies, three mechanisms of the insecticides resistance were reported; one mechanism involved target-site insecticide resistance, and the other two mechanisms involved the increased detoxification of the insecticides. In culex mosquitoes [19] and aphids [20], the resistance against carbamate and organophosphate has been reported, and it is an example of the mechanism of detoxification.

Another example of detoxification is in the two species of flies in which the degradation of insecticides takes place. In specific carboxylesterases, the structural mutations had arisen that used to convert them into inefficient but physiologically sufficient organophosphate hydrolases [21]. The third mechanism of detoxification is the mutation of the target molecule in such a way that the target molecule becomes insensitive to the insecticides. The target molecules that become mutant are: for cyclodienes, the γ-aminobutyric acid (GABA) receptors become mutant; for organophosphates, the acetylcholinesterase becomes mutant; and for synthetic pyrethroids and dichlorodiphenyltrichloroethane (DDT), the voltage-gated sodium channels are becoming mutant [22, 23]. These findings were having some remarkable aspects such as the degradation and sequestration mechanisms, the sodium channels becoming insensitive, and repetition of the same amino acid changes in the orthologous proteins among different species, for example, in the acetyl cholinesterases and GABA receptors. The third aspect of these findings was that within a few years of the first use of insecticide, a small amount of the mutant alleles carry the mutations that have spread among the species. These features have shown that insects have very less options to confer the resistance against insecticides [24, 25].

Genomic technologies are able to investigate the previous intractable mechanisms of the resistance. Genomics also discusses the resistance to the proteinaceous biopesticide crystal toxins of *Bacillus thuringiensis* (Bt toxins) and the traditional chemical insecticides [26]. By the help of genomics, some targeted genes of the nervous system of *Drosophila melanogaster* have been cloned and identified and in a wide range of the pest insects, the resistance associated mutations have been studied [27]. Recently, with the advancement in genome sequencing and annotation techniques, genomes of the insects have been sequenced and annotated and the complex multigene enzyme systems such as the glutathione-S-transferases, esterases, and cytochrome P450 that facilitate the insecticides resistance among the insects have been explored [28]. In 2000, the whole genome of *Drosophila melanogaster* was reported, and after that, the partial and complete genomes of different species of insects have started to publish in the biological databases. In the NCBI database, genome sequences of almost 34 species of the orders Hymenoptera, Coleoptra, Diptera, Hemiptera, and Lepidoptera of the insects are available. These species include the most primitive insect human louse and major medical pests such as *Aedes aegyptii* and *Anopheles gambiae* [28].

#### **5. Genes involved in insecticide resistance in insects**

Recently, insecticide resistance has become a major concern for the control of many insect pest species. This challenging problem has useful solutions in the genome sequencing, transcriptome analysis, and the global quantization of the gene expression of those genes that are involved in the insecticide resistance. One of the most destructive agricultural pests of the world is *Bactrocera dorsalis* (oriental fruit fly), and it is used as a model to examine the genetic mechanisms of the insecticides resistance. For this species, the molecular data of the genes that were identified by homology was very limited. By using the Ilumina Solexa platform of the next-generation sequencing, the whole transcriptome of *Bactrocera dorsalis* was sequenced and the gene expression in the insecticide resistance was explored [29].

Mosquitoes are the major carriers of pathogens, and they are the source of causing infectious diseases among humans such as dengue and malaria, and the control of mosquitoes is the biggest threat as they are resistant to insecticides. In natural populations, the alternative tools for the control of mosquitoes have been implemented and the mechanism of the resistance was studied. A common mechanism of the resistance is the biodegradation of the insecticides by detoxification enzymes; during this mechanism, the changes in the genome of the mosquitoes have been identified except the individual genotyping of the resistance. Particularly, polymorphisms of the detoxification enzymes and the function of the copy number variations (CNVs) have not been examined at the genomic level though they represent strong markers for metabolic resistance. With the use of next-generation sequencing, the genes and polymorphisms associated with insecticide resistance in mosquitoes have been explored. According to a research, 760 candidate genes were sequenced and identified to be the cause of resistance against deltamethrin in the dengue mosquito (*Aedes aegypti*) [30]. The analysis of the CNVs showed the amplification of 41 genes to be associated with the resistance and in the resistant populations, the cytochrome P450 was over transcribed. More than 30,000 variants were detected in the analysis of the polymorphism. By combing the filtering of allele frequency and the Bayesian 55 nonsynonymous variants that were strongly associated in causing the resistance were identified. Both the polymorphisms and the CNVs within the regions were conserved but differed across the continents, which confirm that the changes in the genome causes the metabolic resistance against insecticides are not universal. The novel DNA markers for insecticide resistance were identified, which open the way for tracing the metabolic changes established by the mosquitoes for resisting the insecticides within and among the populations [31].

*Anopheles gambiae* is resistant to the four classes of insecticides, that is, the carbamates, pyrethroids, organophosphates, and organochlorines; that is why the control of the malaria is difficult in Africa. The functional validation of the detoxifying enzymes is lacking in *Anopheles gambiae*, but the expression of the detoxifying enzymes increases in resisting the insecticides. In the resistant *Anopheles gambiae*, the three genes Cyp6p3, Cyp6m2, and Gste2 are upregulated; for these findings and to explore the phenotype of the insecticide resistance, the transgenic analysis was performed using the UAS/GAL4 system. The evidence was reported that the resistance against organochlorine and organophosphate in *Anopheles gambiae* explains the overexpression of GSTE2 in a wide tissue profile. Carbamate and pyrethroid resistance is given by the overexpression of Cyp6p3; in the same tissues, pyrethroid resistance is explained by Cyp6m2. According to a research conducted on 757

*Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects DOI: http://dx.doi.org/10.5772/intechopen.112662*

samples of *Anopheles gambiae*, the mutations in the rdl, ace-1, and kdr gene were detected using sequencing and SNaPshot. In the insecticide resistance in *Anopheles gambiae* populations, the multiple mutations were also detected in the kdrW, ace-1, and A296G rdl alleles [32].

#### **6. Mechanisms of insecticide resistance**

Insecticide resistance is primarily caused by changes in the genes of insects. The genes that are involved in the insecticide resistance include those that encode for detoxification enzymes such as cytochrome P450 (CYP) and glutathione S-transferase (GST), which metabolize and detoxify the insecticides. These enzymes can also have mutations that increase their activity, making the insecticides less toxic. Target-site resistance mechanisms are also driven by mutations in the genes encoding the target proteins of the insecticides. On the other hand, insecticides target specific genes in insects to kill them. These genes are responsible for vital processes such as nerve impulse transmission, muscle contraction, and metabolism. For example, many insecticides target the voltage-gated sodium channels in the insects' nervous system, which are necessary for nerve impulse transmission [11, 16].

Other insecticides target enzymes that are involved in the production of energy in the insects, such as the mitochondrial electron transport chain, making it impossible for the insects to survive. Insecticides also target genes that are responsible for the synthesis of chitin, which is an important component of the insects' exoskeleton and necessary for their survival. It is important to note that the mechanisms of resistance and the target of the insecticides are constantly evolving due to the insects' adaptation to the environment and the insecticides. Therefore, the identification of the specific genes involved in resistance and the genes targeted by insecticides is essential for the development of new insecticides and strategies to control the pests [33]. There are different mechanisms of insecticide resistance, which include target-site insecticide resistance, metabolic insecticide resistance, penetration resistance, and behavioral resistance.

#### **7. Penetration resistance**

The susceptible insects absorb the toxin more quickly than the resistant insects. When the insects' outer cuticle develops the barriers of the slow absorption of the insecticides in their bodies, the penetration resistance occurs. Due to the penetration resistance, insects are protected from a wide range of the insecticides. Along with the other mechanisms of the insecticides, the penetration resistance takes place, and due to the reduced intensity of the penetration, these mechanisms of resistance dominate among insects [34].

#### **8. Behavioral resistance**

The insects that are resistant to insecticides are able to recognize and detect a danger and to avoid the toxin. For various classes of insecticides such as organophosphates, carbamates, organochlorines, and pyrethroids, the behavioral mechanism has been reported [35].

#### **9. Target-site insecticide resistance**

The specific binding site of an insecticide is mutated or modified during the resistance of the target site, due to which the target site becomes incompatible for the activation. In most common pests (such as *Myzus persicae*, *Musca domestica*, and *Drosophila melanogaster*), the mutations occur in the target regions, that is, knockdown resistance to pyrethroids, reduced sensitivity of the sodium channels against DDT, and the resistance against spinosad and subunits like nicotinic acetylcholine receptors for the neonicotinoids [24, 25]. Because of these mutations, the binding of the target region with the insecticides becomes impossible, and this leads to a loss of binding affinity. Moreover, the overproduction of the enzymes occurs in the metabolic resistance, which detoxify or break down the insecticides, leading to the resistance of the pests. Some metabolic enzymes such as hydrolases, cytochrome p450 monooxygenase, and glutathione S-transferase play a major role in the evolution of metabolic resistance. In the wild-type AChE gene (ace), the point mutations were found in the resistant *B. dorsalis*. In some species of the insects, the resistance also arises from the novel variants that represent the genetic changes such as the RNA edited product or alternatively spliced RNA [36].

#### **10. Metabolic insecticide resistance**

Metabolic insecticide resistance, also known as detoxification-based resistance, is a mechanism by which insects are able to detoxify the toxic compounds present in insecticides through the action of enzymes. This type of resistance is becoming increasingly common and is a significant threat to the control of insect pests. According to recent research, metabolic insecticide resistance has been primarily mediated by the activity of enzymes such as cytochrome P450 monooxygenases (P450s), esterases, and glutathione S-transferases (GSTs). These enzymes are able to detoxify the toxic compounds present in insecticides, rendering them harmless to the insect [10].

#### **11. Proteomics- proteins and compounds involved in developing resistance**

One example of metabolic insecticide resistance is found in the cotton bollworm, *Helicoverpa armigera*. Research has shown that this pest is able to detoxify the insecticide deltamethrin through the action of P450 enzymes. Specifically, the study found that the insect had an increased expression of the P450 gene CYP6B8, which was responsible for detoxifying the insecticide. Another example can be found in the red flour beetle, *Tribolium castaneum*. Research has shown that this pest is able to detoxify the insecticide chlorpyrifos through the action of esterases. Specifically, the study found that the insect had an increased activity of the esterase enzyme, which was responsible for detoxifying the insecticide [36–38].

Metabolic insecticide resistance can also be found in the mosquito, *Aedes aegypti*. Research has shown that this pest is able to detoxify the insecticide temephos through the action of GSTs. Specifically, the study found that the insect had an increased activity of the GST enzyme, which was responsible for detoxifying the insecticide. It is important to note that the evolution of resistance in insects is a complex process, influenced by a combination of genetic, biochemical, and environmental factors. To ensure effective control of insect pests, it is crucial to adopt integrated pest management strategies that include the use of insecticides in combination with other control

*Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects DOI: http://dx.doi.org/10.5772/intechopen.112662*

measures such as source reduction, biological control, and the use of alternative treatments such as essential oils. Research shows that metabolic insecticide resistance is a significant problem that is becoming increasingly common. The resistance is primarily mediated by the activity of enzymes such as P450s, esterases, and GSTs, which are able to detoxify the toxic compounds present in insecticides. To effectively control insect pests, it is crucial to adopt integrated pest management strategies that include the use of insecticides in combination with other control measures [30, 31].

The mechanism of the insecticide resistance of some insects is explained here:

#### **11.1 Cockroaches**

In more than half a dozen insect pest species, point mutations in the para sodium channel gene have been linked to knockdown resistance (kdr) to pyrethroids insecticides. In this investigation, we found two novel para variants in five strains of German cockroaches with high levels of resistance to kdr. The first intracellular linker, which joins domains I and II, contains the two alterations, which change glutamic acid (E434) to lysine (K434) and cysteine (C764) to arginine (R764), respectively. Closest to domain I is E434K, which is found near the beginning of the linker. C764R is found near the end of the linker (closest to domain II). One of the resistant strains has two further mutations, one from proline (P1880) to leucine (L1888) and another from aspartic acid (D58) to glycine (G58). The four mutations are exclusively seen in the most resistant individuals of a particular strain, and they coexist with the previously discovered leucine to phenylalanine (L993F) kdr mutation in IIS6. These findings imply that these mutations may be in charge of the German cockroach's high levels of knockdown resistance to pyrethroids pesticides [39, 40].

#### **11.2 Head lice**

*Pediculus humanus capitis*, often known as the human head louse, is a bloodsucking ectoparasite that primarily affects kids in both industrialized and developing nations. Permethrin is the primary active component of chemical pediculicides, which are the first line of defense. Despite the prolonged usage of these products, no studies have been conducted to determine if head lice in Honduras are resistant to insecticides. Knockdown resistance (kdr), the most prevalent mechanism in head lice, is caused by two point mutations and the corresponding amino acid substitutions, T917I and L920F, in the voltage-sensitive sodium channel (VSSC) [41]. The most significant contributing factor to the rise in head lice infestations worldwide may be pyrethroids resistance [42, 43]. Knockdown resistance (kdr), which reduces an insect's nerve sensitivity, is a property of lice resistant to pyrethroids and is brought on by single nucleotide point mutations (SNPs) in the para-orthologous voltagesensitive sodium channel (VSSC) gene. It is well recognized that resistance is caused by the key amino acid substitutions T917I and L920F, which are found in domain II [44]. The locations of the housefly VSSC's amino acid sequence revealed that the mutations T929I and L932F, which have been linked to permethrin resistance, were expressed (rather than in the head louse amino acid sequence). Additionally, it has been shown that this group of mutations coexists as a resistant haplotype; when T197I was produced in *Xenopus oocytes*, either alone or in combination, it effectively inhibited permethrin sensitivity. The T917I amino acid change is relevant to pyrethroid resistance via the kdr-type nerve insensitivity mechanism and can be employed as a molecular marker for resistance detection [45].

#### **11.3 Fruit fly**

Cyclodiene and phenylpyrazole insecticides affect the GABA-gated chloride channel component that the resistance to dieldrin gene, or Rdl, encodes. By genetically mapping cyclodiene dieldrin resistance in *Drosophila melanogaster*, the gene was first identified. The change from Ala301 to Ser, one amino acid, caused the 4000-fold resistance. A wide variety of resistant insect species' Rdl orthologs were later found to contain the same alteration. In a research, a duplication at the Rdl gene in *D. melanogaster* was discovered. Rdl is present in two copies, one of which is WT and the other of which has two point mutations: An Ala301 to Ser resistance mutation and a Met360 to Ile substitution. Individuals with this duplication had lower temperature sensitivity, altered RNA editing linked to the resistant allele, and intermediate dieldrin resistance compared to single copy Ser301 homozygotes. This genomic rearrangement is caused by ectopic recombination between Roo transposable elements. By building a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser301 mutation into an Ala301 background, the duplication phenotypes were confirmed. In most cases, gene duplications increase the amount of gene product generated, which has a considerable impact on the evolution of pesticide resistance. However, in this instance, duplication of the Rdl target site results in permanent heterozygosity, offering a rare opportunity for adaptive mutations to accumulate in a single copy without removing the essential gene's innate function [46].

#### **11.4 Mosquito**

The environmental changes in nature and the adaptive genes are easily identifiable; *Culex pipiens* mosquito's resistance to organophosphorus pesticides provides a useful model for analyzing the fitness cost of resistance genes and their origin. This resistance is caused by two loci, the super-locus Ester and the locus Ace.1, each of which contains a number of resistance alleles. According to population surveys, the fitness costs of various resistance genes and even resistance alleles at the same locus vary. The consequences of these resistance genes on various fitness-related variables are being investigated in order to better understand this fitness cost and its unpredictability. The impact of three resistance alleles such as Ester4, Ester1, and Ace.1R on paternity success relative to susceptible males and relative to one another in the research using competition trials between two males for accessing a single female were examined. The impact of susceptible and resistant female genotypes on male mating success was eventually examined. The strains utilized in this investigation have a common genetic history. Males who competed against any of the resistant males had a mating advantage, indicating a high cost of resistance genes for this feature. Regardless of the genotype of the female, resistant male had the same paternity success rate when competing against susceptible males [31, 32, 38, 47].

#### **12. Pathways involved in metabolic resistance**

Xenobiotics are detoxified by enzymes into a less or nontoxic compound, resulting in the formation of a more suitable form of metabolite for rapid removal from the body. Insects having resistance metabolize these insecticidal compounds faster due to presence of enzyme with increased catalytic rate and in higher quantities because

#### *Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects DOI: http://dx.doi.org/10.5772/intechopen.112662*

of increased amplification and transcription of their genes. There are two phases of detoxification: phase I (primary), consisting of oxidation or hydrolysis, and phase II (secondary), consisting of conjugation reactions of products of phase I with different endogenous compounds, like glucuronic acid or glutathione, facilitating their subsequent dissolution and excretion from all over the body [48–51]. Sequestration is also an important mechanism of defense that has been adopted by insects to tolerate these xenobiotics, in addition to such processes of detoxification that are based on cleavage and excretion of insecticides by using enzymes. This strategy involves selective and specific uptake, transportation, and storing of secondary metabolites from the plants on which they are feeding. These metabolites provide them resistance against the insecticides, interfering with their physiological mechanisms [52, 53]. One of the examples of such mechanisms is hematophagy, found in mosquitoes. It could be probably a way of secondary adaptation in which they obtain food of high quality in order to maintain egg production [54].

The enzymes that are majorly involved in xenobiotics detoxification in living organisms are synthesized by transcription of members of large families multigene complexes of enzymes like oxidases, esterases, and glutathione transferases (GSTs).

#### **12.1 Esterases**

Esterases belong to a large group of enzymes that catalyze phase 1 reactions, which can metabolize a large variety of endogenous and exogenous substrates. Their role in detoxification of insecticide metabolites is well reported, and they have been shown to act against a wide range of chemical compounds, including organophosphates, pyrethroids, and carbamates [48]. Studies have shown their probable involvement in resistance against Bt toxin [55] and even against neonicotinoid [56]. Insecticide compounds can be detoxified through enzymatic cleavage or sequestration. Insecticides esters are hydrolyzed into their corresponding alcohols and acids by the Esterases and are excreted from the insects' body more easily due to their increased solubility. Insecticides can also be sequestered by Esterases so that the availability of toxic molecules is no longer possible for interacting with the target proteins [57–59]. Esterases are linked to insecticide resistance, due to some qualitative or quantitative or both types of changes in many species of insects, causing the enzymes' overproduction or their structures modifications [48]. Esterases are overexpressed due to upregulation of their genes or amplification or both. One of the most studied examples of detoxification of insecticide through gene amplification is seen in the green peach aphid *Myzus persicae*, which involves the overproduction of a specific enzyme carboxylesterase (Hemiptera: Aphididae) [60–63]. Such amplified esterases have also been seen in mosquitoes of the genus Culex, associated with insecticide resistance (Diptera: Culicidae) [19, 64, 65] and some other species, like the brown planthopper *Nilaparvata lugens* (Stal) (Hemiptera: Delphacidae) [66]. In some species, like *Aphis gossypii* Glover (Hemiptera: Aphididae) or B-biotype *Bemisia tabaci* (Gennadius) (Hemiptera: Aleyrodidae), the expression of enzymes esterases is increased due to increased levels of transcription, due to corresponding gene upregulation [67, 68]. Esterases are also involved in changing the structure of enzymes that are involved in enhanced ability of insects to metabolize the compounds of insecticides. "Mutant ali-esterase theory" was presented by scientists based on this type of mechanism described in the housefly *Musca domestica* (Diptera: Muscidae) for the first time [69]. The insects with resistance exhibited a decreased activity of esterase apparently, as compared to susceptible compounds, resulting due to structural modifications in the

enzyme facilitating the process of hydrolysis of the metabolite of insecticide, but it reduced or prevented the hydrolysis of the molecule used for determination of the esterase activity. This mechanism of resistance was considered to be based on substitution of two amino-acid (Gly137Asp and Trp251Leu) in houseflies as well as in many other insect species belonging to the order of Diptera [22, 23, 70].

Other mechanisms like chromosomal rearrangements or demethylation also effect the overproduction of esterase. Mechanisms of demethylation can lead to gene silencing and subsequent reduction of levels of esterase among E4 populations [71], whereas no correlations have been seen between esterase activity and methylation levels in the esterase variant of FE4 [61]. In some Italian populations of aphid species, autosomal rearrangements of same type for the FE4 isoforms of esterase have been reported but only in those with low activity of esterase, showing that resistance due to esterase and translocation are not correlated always [62, 72].

#### **12.2 Monooxygenases**

Another class of enzymes involved in metabolism of Xenobiotics is microsomal oxidases or mixed function oxidases (MFOs). These are enzymes of phase 1 reactions and are also involved in metabolism of endogenous metabolites like fatty acids, pheromones, or hormones. These enzymes can convert hydrophobic molecules to hydrophilic substances so that they can easily be eradicated from the body. Their major localization is in digestive tract [37, 73, 74]. Microsomal oxidases are Cytochrome P450 monooxygenases (P450s) that are from the group of enzymes composed of heme thiolate proteins. They exhibit a characteristic peak of absorbance at 450 nm when they are in a reduced form and complexed with molecule of carbon monoxide. The reactions catalyzed by these enzymes involve the transfer of one atom of molecular oxygen to a substrate and the reduction of the second atom of oxygen to form water. This process needs the transfer of at least two electrons, which are provided by NADPH cytochrome P450 reductase [73, 75]. P450s possess a large variety of enzymes that are highly specific for substrate and can catalyze various reactions like hydroxylation, epoxidation, desulfurization, O-dealkylation, or N-dealkylation. They play a major role in interactions between plants and insects and metabolizing many insecticides like organophosphates, carbamates, neonicotinoids, and pyrethoids [50, 76–79]. The enzymes of P450 family are named by abbreviation CYP with an Arabic number of the respective family, a capital letter designating the subfamily with an Arabic numeral designating the individual protein. Every each has its own gene to be coded. More than 600 P450 genes have been characterized from insects, and it has been found that genes of families CYP6, CYP4, CYP12, and CYP9 are associated with resistance in insects against insecticides (**Figures 1** and **2**) [48, 73, 74]. The MPOs are majorly found in the midgut, Malpighian tubules, and fat bodies of insects. The housefly is the candidate whose MPOs system has been extensively studied [80]. Studies have reported that higher concentrations of P450s and an increased activity of monooxygenases are found in resistant insects. Overexpression of such activities occurs as a result of upregulation of their genes, that is mediated by the modifications of regulatory elements [73]. They have also shown amplification of genes or qualitative modifications in other studies [78, 81–83]. Some type of insecticides can also be activated by enzymes of insect P450 system. One of the example is the formation of phosphate (P=O) from phosphorothioates (P=S). This causes an increased 330 potency for inhibiting acetylcholinesterase by a magnitude of 3 or 4 orders. The synthesis of juvenile hormone, pheromone components, and ecdysone also needs involvement of P450s [84].

*Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects DOI: http://dx.doi.org/10.5772/intechopen.112662*

#### **Figure 1.**

*Metabolism of xenobiotics by cytochrome P450 - Anopheles gambiae (malaria mosquito).*

Aerobic organisms are characterized by the presence of a diverse group or family of enzymatic proteins named Glutathione transferases (GSTs), which are found ubiquitously. They play a major function in the detoxification of xenobiotic as well as endogenous compounds. They are also found to be associated with synthesis of hormones, intracellular transport, and protection in contradiction of oxidative stress [85]. GST enzymes are involved in catalyzing the conjugation reactions of reduced glutathione with electrophilic molecules or substrates as well as in sequestrating substrate. This results in increased hydrophilicity or water solubility and decreased toxicity of reactive molecules and in turn facilitates their removal or excretion from the body. Specifically, they catalyze conjugations by facilitating nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione (GSH) on electrophilic centers of a range of xenobiotic

#### **Figure 2.**

*Glutathione metabolism - Drosophila melanogaster (fruit fly).*

compounds, including insecticides or acaricides [86] and various plant toxins [76]. This results in the conversion of xenobiotics into derivatives of mercapturic acid, which are more soluble and easily excreted out from the insect body [87, 88]. Species of free oxygen radicals are formed in insects by the action of pesticides that are highly toxic and can be removed with the help of these GSTs. They also help in metabolizing insecticides through their reductive dehydrochlorination [89]. In insects, there are two groups of GSTs: cytosolic and microsomal. They are classified on the basis of their occurrence in the cell. Only cytosolic GSTs have been reported to be involved in insecticides metabolism. They play an important role in developing resistance against some insecticides like pyrethroids and organophosphates. In mosquitoes and houseflies, resistance against DDT has been eveloved due to a DDT dehydrochlorinase GST [88]. The full extent of family of this enzyme has been revealed in genomes of the *Drosophila melanogaster* Meigen and *Anopheles gambiae* Giles [88]. Generally, the quantity of enzyme is increased due to either overexpression of gene or its amplification, which results in enhanced resistance on the basis of GSTs [90, 91]. Insecticides can also be sequestered by GSTs that provide the insects protection against the toxicity of these insectcides, for example, pyrethroid [83].

*Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects DOI: http://dx.doi.org/10.5772/intechopen.112662*

#### **12.3 Pgp pumps**

Pgp pumps are transporters composed of P-glycoprotein (Pgp) that are integral membrane proteins and belong to the ATP-binding cassette (ABC) superfamily, which utilizes the energy produced from ATP breakdown and translocates different metabolites as well as xenobiotics across the cell membranes [92]. This type of mechanism has been majorly observed in fungi and bacteria for developing resistance against antibiotics [93], but very little work has been reported on it regarding insects. Only recently, these ABC transporters have been found in insects as a supposed mechanism that can contribute in resistance by facilitating the efflux transport mechanism of insecticides as well as their compounds or metabolites that are derived from phase I and II reactions [94–98]. ABC transporters can produce resistance in insects through different modes like quantification of protein or transcript and by synergistic mechanisms of ABC inhibitors [94, 99]. Furthermore, in different lepidopteran species, a mutant allele has been discovered that confers resistance to the pore-forming Cry1Ac toxin from *Bacillus thuringiensis* (Bt) by a mechanism that is not related to toxin extrusion, but because it causes the loss of Cry1Ac binding to membrane vesicles [100, 101].

#### **13. What is systems biology?**

The study of the relationships and behavior of biological entity components such as molecules, cells, organs, and organisms is known as systems biology. Individual roles are played by microbes, plants, animals, and entire ecosystems in the natural world, which is a complex system of interconnected pieces. The investigation of living creatures is approached comprehensively in systems biology. It studies how diverse biological creatures interact at different sizes. Every person, for example, is a system. The system includes our organs, tissues, cells, and the components they are formed of, as well as bacteria and other creatures that dwell on our epidermis and in our digestive system [102].

Computational and mathematical analysis and modeling are important to systems biology. It gathers data from a wide variety of biological sciences and technologies known as "-omics" by researchers. Among these "omics" are genomics (the study of whole gene sets in an organism) and proteomics (the study of all the proteins in a cell, tissue, or organism). The emphasis in these fields is on describing and measuring the biological molecules that underpin how organisms are produced, operate, and live [103].

#### **14. What is a significant role of systems biology in causing insect resistant?**

Insecticide resistance is regarded as a typical similar pattern of microevolution, in which a powerful selection agent is given to a large natural community, resulting in a shift in the frequency of alleles conferring resistance. While numerous pesticide resistance variations have been identified at the gene level that was in term of systems biology, they are usually single genes with a big influence seen in highly resistant insect pest. With *Drosophila melanogaster,* many polymorphisms have been involved in DDT resistance; however, only Cyp6g1 locus has already been proven to be meaningful to field populations. They uncover DDT-associated polygenes using genome-wide association studies (GWAS) and assess their

adaptive importance using selective sweep analysis. As a result, they validate two DDT resistance loci. This was considered as the significant role of system biology in causing insect resistant [104].

#### **15. What are the main pathways involved in insects resistance?**

The two major pathways involved in insecticide resistance were metabolic resistance and target-site resistance. Metabolic resistance is a typical defense strategy that relies on enzymatic mechanisms to protect the insect by detoxifying/sequestering pesticide compounds. In order to overcome the potential toxicity of the plants they feed on, the enzymes involved are those that insects have evolved as support against naturally occurring plant poisons (study will focus) such as alkaloids, terpenes, and phenols. This might explain the modernization of metabolic resistance to a wide range of insecticides, many of which have direct or indirect botanical origins. Enzymes may detoxify xenobiotics into a nontoxic chemical and/or a form that is more suited for fast removal from the body [105].

Resistant insects metabolize the pesticide quicker because they have enzymes with a better catalytic rate, or because they have more enzymes as a result of enhanced transcription or gene duplication. Detoxification can be separated into two phases: phase I (primary) activities involving hydrolysis or oxidation, and phase II (secondary) processes involving coupling of phase I results with endogenous molecules such as glutathione and eventual elimination from the body. In addition to such enzymatic cleavage and excretion-based detoxification methods, sequestration is a significant defense mechanism that certain insects have evolved to withstand xenobiotics [67].

This is a typical phenomenon in insect herbivores that involves the precise and selective absorption, transport, and storing of secondary metabolites from plants in order to avoid interference with the insects' physiological processes. Such behavior has been seen in mosquitos, where hematophagy is most likely a subsequent adaption to get high-quality food for egg formation. Members of vast multigene families of isoenzymes, oxidoreductases, and GSTs transcribe the enzymes involved in xenobiotic detoxification in living organisms [106].

On the other hand, target-site resistance was explained as the pesticide's target site of action in the insect that can be genetically engineered to inhibit the insecticide from bonding or interacting at the site of action, lowering or eliminating the insecticide's pesticidal impact. During target-site resistance, an insecticide's particular binding site is transformed (mutated) and/or removed, rendering the target site unsuitable with activation. Most frequent insect (*Myzus persicae*, *Musca domestica*, and *Drosophila melanogaster*) target areas are mutated, including subunits such as cholinergic acetyl cholinergic receptor (nAChRs), knockdown resistance (KDR), and others. Insecticides are not able to bind inside the target area as a result of these changes, resulting in a reduction of binding affinity [107].

#### **16. Which mechanism of resistance affects the behavior of the insects?**

Metabolic resistance serves as the most common mode and frequently poses the most difficult barrier. Insects break down pesticides using their internal enzyme systems. These enzymes may be present in larger concentrations or in more effective forms in resistant strains. It was also explained by the case study of P450 gene in *House Flies* [108].

#### *Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects DOI: http://dx.doi.org/10.5772/intechopen.112662*

Insects may employ a variety of metabolic processes to avoid the fatal effects of pesticides. Increased cytochrome P450 detoxification, for example, is known to play a key role in many insect species. P450s' constitutively elevated overexpression and induction are hypothesized to somehow be responsible for enhanced levels of pesticide detoxification. However, unlike continuously upregulation P450 genes, whose regulation connection with pesticide resistance has been well explored; P450 induction in insecticide resistance is less well understood. The current work focuses on the identification of particular P450 genes that are activated in permethrin-resistant house flies in response to permethrin treatment. As a result, Permethrin administration co-upregulated the expression of three P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, in permethrin conferring resistance ALHF house flies in a period and dosedependent way. The protein sequences among these 3 P450s from resistant ALHF as well as vulnerable aabys and CS house flies were found to be similar. CYP4D4v2 and CYP6A38 were found on autosome 5, correlating to the association of P450-mediated resistance in ALHF, while CYP4G2 was found on autosome 3, where the key insecticide susceptibility factors for ALHF had been mapped, but no P450 genes had been reported previously to this investigation.

This study provided the first direct proof that numerous P450 genes are co-upregulated in permethrin-resistant house flies via the induction process, which boosts total P450 gene expression levels in resistant house flies. This research provides new information on the functional importance of P450 genes as they react to insecticide therapies, detoxification of insecticides, insect adaptation to their atmosphere, and the evolution of insects [108].

#### **17. What is the role of protein-protein interaction pathway in insect resistance according to system biology?**

At the moment, the problem of resistance is not fundamentally solved since the development speed of new insecticides cannot keep up with the progression speed of resistance, and there is a lack of knowledge of the molecular mechanism of resistance.

Researchers used literature mining and the String database to identify seed genes and their interacting proteins involved in the biological mechanism of pesticide resistance in *Drosophila melanogaster*. They discovered 528 proteins molecules and 13,514 protein-protein interactions. String and Pajek built the protein interaction network, and we looked at topological features like degree centrality and eigenvector centrality. KEGG pathway enrichment analyses revealed an enrichment for proteasome complexes and drug metabolism of cytochrome P450. This is the first time that the pesticide resistance in molecular level mechanism of *D. melanogaster* has been investigated using network biology methodologies and tools, and it can provide a bioinformatic basis for further understanding of insecticide resistance mechanisms [108].

So, systems biology plays a very important role in the insect resistance against insecticides and different chemicals such as DDT and permethrin. From systems biology, not only the identification of genes was done, but also the protein-protein interactions were found out, which were responsible for insect resistance.

#### **Author details**

Rabbiah Manzoor Malik1,2,3\*, Sahar Fazal3 , Narjis Khatoon3 , Muneeba Ishtiaq3 , Saima Batool3 and Syed Tauqeer Abbas1,2

1 Wah Medical College, Wah Cantt, Pakistan

2 National University of Medical Sciences, Rawalpindi, Pakistan

3 Capital University of Sciences and Technology, Islamabad, Pakistan

\*Address all correspondence to: rabbiahmanzoor@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Perspective Chapter: Genomics, Proteomics, and System Biology of Insecticides Resistance in Insects DOI: http://dx.doi.org/10.5772/intechopen.112662*

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Section 3

## Insect Pest Control by Pheromones Plant Extract

### **Chapter 6**

Potency of Neem, *Azadirachta indica* L. (A. Juss) Leaf Aqueous Extract Insecticide against White Mango Scale, *Aulacuspis tubercularis* Newstead (Homoptera: Diaspididae) Infesting Mango (*Mangifera indica* L.)

*Temesgen Fita, Emana Getu, Mulatu Wakgari and Kebede Woldetsadike*

#### **Abstract**

This study was conducted to evaluate the potency of aqueous *A. indica* leaf powder extract (LPWE) against *A. tubercularis* infesting mango. Field experiments were conducted in Ethiopia at two experimental sites, western Oromia. Randomized Complete Block design was laid in four replications with four blocks consisting of sixteen treatment plots. The treatments were 0.05, 0.1 and 0.15 mg/ml spray concentrations made from *A. indica* LPWE. The treatments were applied 3 times at 10-day interval and the mortality count was carried out 10 days after 1st, 2nd and 3rd treatment applications. The results of the study confirmed that among the tested three different concentration of *A. indica* LPWE 0.15 mg/ml concentration significantly (p < 0.05) reduced the population of sessile *A. tubercularis* at both experimental sites. The results also indicated that male adults and nymphs were more affected than females. The population of sessile *A. tubercularis* significantly decreased as the concentration of *A. indica* LPWE increased in comparison with the check plots. Thus, the use of *A. indica* LPWE at high concentrations led to a notable population reduction of sessile *A. tubercularis* and its effects. Hence, the use of *A. indica* LPWE can be recommended for *A. tubercularis* management as part of integrated pest management.

**Keywords:** *Azadirachta indica*, aqueous extract, *Aulacaspis tubercularis*, botanical insecticide, management, nymphs, potency

#### **1. Introduction**

Mango is one of the most widely cultivated fruit crops in Ethiopia, preceded only by bananas in terms of economic importance. Most of the mango productions come mainly from the southwestern, western, central rift valley, and eastern parts of Ethiopia [1] The annual mango production in Ethiopia is 151,331.24 t with an area coverage of 20,783.92 ha, and its production is 7.28 tones ha−1 [2] which accounts for about 0.18% of the world production. Mango is attacked by multiple arthropod insect pests among the damage inflicted by the white mango scale, and *Aulacaspis tubercularis/mangiferae* Newstead (Homoptera: Coccoidea: Diaspididae) is the most important insect pest of mango [3, 4].

This insect is a cosmopolitan pest throughout the globe regardless of where mango is produced [5, 6]. *A. tubercularis* is a tropical species believed to have originated from tropical Asia [7]. White mango scale *A. tubercularis* global dispersal could have taken place through the movement of plant materials [8]. This insect pest was first identified in Ethiopia in 2010 invading private commercial mango plants in East Wollega Zone, Oromia Regional State, western Ethiopia [3] from where it was dispersed to distinct mango-growing parts of the country [4]. At the climax level of infestations, *A*. *tubercularis* generates production-dropping covering from 50 to 100% [9].

Utilization of the synthetic chemical insecticide to control insect pests causes many environmental problems such as destabilizing the biodiversity and trophic level of the ecosystem, harmful effects on animals, human health, and the beneficial insects [10]. It has also been observed that though the chemical industry is aware of the environmental effect of the misuse of pesticides, they are not giving due regards to promotion of ecologically sound practices that will enhance sustainability in agricultural production [11]. Reducing chemical pesticide use has become globally shared issues in several countries and become a major issue in public policies due its negative impacts on the environment and on human health [12]. Homemade botanical insecticides are widely used by subsistence and transitional farmers in low-income countries. Their use is often driven by the limited availability or cost of commercial pesticides. Homemade botanical insecticides are often recommended by agricultural extension services and some development organizations. However, this could be questioned because scientific evidence of their efficacy and safety may not be available or accessible [13].

One of the most economically important potential plants to become a vegetative pesticide is the neem tree*, Axadirachta indica L.* (A. Juss.), which contains azadirachtin that is accumulated on the leaves and specifically in the seeds [14]. Neem-based pesticides play a vital role in pest management and hence have been widely used in agriculture. Mostly all parts of the neem tree like the leaves, bark, flowers, seeds, and fruit pulp are used in the powdery or in the extract form like leaf extract, kernel extract, cake extract, oil spray, etc. [15]. Neem *A. indica* is a plant having evergreen leaves all year round; however, the fruits and seeds can only be available once a year. In recent times and following the isolation of azadirachtin, the major active compound, that is mainly responsible for the insecticidal activity of neem, the use of neem-based insecticide has increased in the last 30 years, and it is currently the most successful botanical pesticide in agricultural use worldwide [16, 17]. Azadirachtin acts as an antifeedant, repellent, and repugnant agent and induces sterility in insects by preventing oviposition and interrupting sperm production in males [16, 18, 19]. When comparing neem oil formulations with synthetic larvicides, it is costly but neem oil was more effective than the synthetic one for preventing pest resistance [20]*. A. indica* seed powder water

*Potency of* Azadirachta indica *L. Leaf Aqueous Extract Insecticide against* Aulacaspis*… DOI: http://dx.doi.org/10.5772/intechopen.110256*

extracts at 0.15 concentration have a better impact on knocking down the population of *A*. *tubercularis*, and it can potentially be used for the management of the newly emerging and inflicting mango pest, *A. tubercularis* [9]. Extract of neem, *A. indica*, works in a way of interfering with the reproduction, growth of insect, and behavior in the forms of repellent, attractant, antifeedant, and poisoning larva and imago, either as a pesticide or as a contact poison [21]. However, its mechanisms of action still unclear and remain to be clarified, especially in relation to the neurophysiological and the possible longterm activities [22]. It is eco-friendly and has nontoxic actions, and the peculiar mode of action as well as its broad-spectrum activity over chemical pesticides has offered many advantages to beneficial organisms. Like to *A. indica* seeds, *Azadirchatin* is also present in *A. indica* leaf with moderate concentrations, and its availability all-round the year make to give focus to the leaf. Therefore, this research aimed on assessing the potency of *A. indica* leaf powder water extract (LPWE) against *A*. *tubercularis* as a contribution to the control of this mango damaging insect.

#### **2. Materials and methods**

#### **2.1 Study area description**

The insecticidal potency test of neem *A. indica* leaf powder extract was carried out during the cropping season of March to May 2018 at Uke Kersa Farmers Administrative of Guto Gida district and Arjo Gudatu Farmers Administrative Kebele, Digga District of East Wollega Administrative zone of Oromia Regional State, western Ethiopia. The selected two study areas represent mango-growing areas of western Oromia Regional State. The ecological zones of the study areas are lowlands (wet kolla), (**Table 1**) with slight altitudinal differences, and both sites are suitable for mango production (**Figure 1**).

#### **2.2 Research materials preparation**

Deep green leaves of neem trees piled up from Dire Dawa city administration, east Ethiopia. The collected leaves were cleaned exhaustively by clean water at its crisp stage to avoid any waste matter or any accumulations amassing materials from the collected leaves, which may reduce the potency of the final *A. indica* leaf extract hurt the apparatus during rectification. The then under shade, plastic sheets were placed on wooden benches and the cleaned leaves were thoroughly distributed *on the sheets* for good air circulation until absolute green drying. The dried leaves were squashed and stocked in cloth sack. In accordance with the [10, 19] procedures, the crushed *A. indica* neem leaves were ground gently to make fine powder using coffee grinder (Coffee and Spice Grinder 220-240 V 50-60HZ', model no. SZJ-830 'S SAYONA Patirrier DELUXE). The ground *A. indica* leave screened by 1 mm<sup>2</sup> mesh wire to obtain a fine particle. Obeying the [23] procedures, with some adjustment, the readymade leaf powder was measured at an amount of 0, 50, 100, and 150 mg and was added into four plastic bucket each containing 15 l of pure water to make up a 0, 5, 10, and 15% solvent, respectively. Then the neem leaf powder extracts in each bucket were mixed gently and vortexed very well. The readymade solvents were stored in the laboratory at an ambient temperature of 27 ± 2°C throughout the period of the study. The leaf extract was filtered by using a muslin bag cloth. To stick the pure filtrate of neem powder water extract on


*NB: G.G. = Guto Gida; Min. =minimum; Max. =maximum; Aver. = average; m.a.s.l. = meter above sea level; mm = millimeter; RH = relative humidity.*

#### **Table 1.**

*Argo-ecological and coordinates of the study area [Source: Ethiopian Meteorology Agency (EMA)].*

#### **Figure 1.**

*The log concentration and probit mortality regression line graph for* A. indica *LPWE treatments against sessile WMS (nymphs, adult females and males) after 1st, 2nd, and 3rd round treatment at Arjo farmers' association administration experimental site.*

the leaf surface of the mango plants, soap with no detergent was added at the rate of 1 ml/liter as an emulsifier [24].

#### **2.3 Study design and treatment application**

The experiments were conducted during the cropping season of April to May 2018 at two experimental sites of Arjo Gudetu and Uke Kersa farmers administration of Diga and Guto Gida districts, east Wollega zone of Oromia Regional State, western Ethiopia. Mango plants of similar size and age were considered for the experiment. For the last 2 years, no pesticides were applied to the mango trees. Randomized

*Potency of* Azadirachta indica *L. Leaf Aqueous Extract Insecticide against* Aulacaspis*… DOI: http://dx.doi.org/10.5772/intechopen.110256*

complete block design (RCBD) was laid in four replications with four blocks consisting of 16 mango plants as treatment plots. A plot consists of one mango plant. Spacing between plants and rows was 7 m and 8 m, respectively. The block size was 224m<sup>2</sup> having four mango plants. Separate three treatment concentrations were made ready from neem leaf powder extracts. The treatments were 0.05, 0.1, and 0.15 mg/ml concentration, and the check plots (neutral water) were used as a negative control for comparison. The *A. indica* aqueous leaf powder extracts were sprayed in four replications.

The concentrations were calculated using the following formula (Eq. (1)):

$$\mathbf{C}\_1 \mathbf{V}\_1 = \mathbf{C}\_2 \mathbf{V}\_2,\tag{1}$$

where C1 and C2 represent initial and final concentration, respectively, and V1 and V2 represent initial and final volume, respectively [25].

After the *A. tubercularis* population approximately reached the economic injury level (EIL) (above five clusters population per leaf), the application was commenced and continued for three rounds at an interval of 10 days. A randomized complete block design (RCBD) was laid out for a field experiment in four replications. The mortality count of sessile *A. tubercularis* was recorded at after 7 days of the 1st, 2nd, and 3rd treatments application. A manually calibrated 'Knapsack Sprayer (Jacto16 HD400 Sprayer Thailand made) with a capacity of 16 l was used for treatment application. Accordingly, for complete spray, 12 l of water was used for each plant. Even though there is no established economic threshold level (ETL) of *A. tubercularis*, assuming that when the pest population reached the economic injury level (EIL), treatment application was commenced and repeated every 10 days from 20 April to 20 May 2018 for three rounds. Spraying of the neem leaf extract was carried out in the afternoon at 3:30 pm to reduce the loss of the chemicals due to evaporation.

#### **2.4 Data collection**

Twelve leaves were plucked from the sprayed mango trees from the middle, lower, and top canopies at four cardinals of each sampled tree 10 days after treatment application (DATA). The collected samples were reserved in paper bags and labeled accordingly. Samples were then put into plastic bugs, transported within a day to the School of Veterinary Science Laboratory, Wallaga University, and reserved at room temperature of 27 ± 2°C. On the next day, the sampled mango leaves and the number of deadly nymphs and adults of *A. tubercularis* were counted under a dissecting stereomicroscope*.* The reduction percentage of the *A. tubercularis* population was the basis of the evaluation of the tested botanical aqueous extract. The counted dead *A. tubercularis* insect nymphs and adult data were converted into corrected percent mortality. Pre- and post-spray counts of the nymphs and adults per leaf were also recorded from the sampled leaves, and the decrement in nymph and adult numbers by the application of aqueous *A. indica* LPWE (efficacy %) was computed based on the [26] equation (Eq. (2)).

$$\text{Efficiency} \left( \text{corrected} \right) \% = \left( 1 - \frac{\text{n in Co before treatment} \ast \text{n in T after treatment}}{\text{n in Co after treatment} \ast \text{n in T before treatment}} \right) \ast 100 \tag{2}$$

where n = Insect population, T = treated, and Co = control.

Due to probable phytotoxicity, any change in the color and texture of mango leaves treated with neem leaf powder extract was noted. The outcome of the deadly nymphs and adults count were expressed in percentage (D.P. %) with correction factor for control/check plots using Abbott's formula [27] as (Eq. (3)).

$$\text{Mortality} \text{(corrected)} \%= \left( 1 - \frac{\text{n in T after treatment}}{\text{n in Co after treatment}} \right) \text{\*100} \tag{3}$$

where n = Insect population, T = treated, and Co = control.

Nymph and adult stages of *A. tubercularis* were regarded as extinct/lifeless if there is alter in color (darkened or dusky), desiccated and or vacant, and motion-less of organs/protuberance when stroked/patted with a feathery brush.

#### **2.5 Statistical analysis**

The collected data were subjected to Statistical Analysis System (SAS) software version 9.0 [28]. The mixed procedure repeated-measure with restricted maximum likelihood (REML) models were employed for statistical analysis of variation between experimental units [29]. Tukey's honestly significant difference (HSD) method was used for mean separation at P < 0.05 level of significance. The *LC*<sup>50</sup> and *LC*<sup>95</sup> of the treatment were calculated by Finney's probit *data* analysis method *using an* MS Excel worksheet [30]. The confidence limits deployed on the treatment concentrations and the probit-mortality of *A. tubercularis* resolved by the logistic regression method [31].

#### **3. Results**

#### **3.1 Potency of** *A. indica* **LPWE against sessile** *A. tubercularis* **nymphs and adults**

The results of the study revealed that mortality of *A. tubercularis* nymphs and adults raised significantly (p < 0.001) with an increase in the concentration of *A. indica* leaf powder extract. The mortality of nymphs and adults of *A. tubercularis* due to different concentrations of *A. indica* LPWE at Uke Kersa and Arjo Farmers' Administrative Kebele experimental sites is shown in **Table 2**. At both experimental sites, the highest mean percent mortality rate of sessile *A. tubercularis* (nymphs, adult males and females) were (±SE) 72.28 and 73.16 at Uke Kersa and Arjo Farmers experimental sites, respectively, during the 3rd round treatment applications with 15% aqueous *A. indica* LPWE treatment concentrations. Moreover, the highest mean percent mortality was recorded on nymphs with 15% *A. indica* LPWE during the 3rd round of treatment with mortality of 87.18 and 89.06 at Uke Kersa and Arjo Farmers experimental sites, respectively. However, the least mean percent mortality was scored with a 5% treatment concentration, during the 1st round of treatment application with mortality of 30.81 and 32.58 at Uke Kersa and Arjo Farmers experimental sites, respectively. Under control treatments, natural deaths were also observed due to hot environmental conditions.

The result of the study depicted that increased mortality of sessile *A. tubercularis* increased with an increase in the concentration of aqueous *A. indica* LPWE


*Potency of* Azadirachta indica *L. Leaf Aqueous Extract Insecticide against* Aulacaspis*… DOI: http://dx.doi.org/10.5772/intechopen.110256*

*\* Within a column means go along with alike letter (s) are not notably unlike from each other (P < 0.05) by Tukey's studentized range test (HSD).*

#### **Table 2.**

*The potency of* A. indica *LPWE treatment application on* A. tubercularis *nymphs and adults during consecutive three-round treatment application (TA) and days after treatment application (DATA) at Arjo Gudetu and Uke Kersa farmers experimental sites.*

treatments application. Female *A. tubercularis* were more tolerant than males and nymphs to all levels of aqueous *A. indica* LPWE treatments concentration. From the result of the experiment, it was clearly observed that the 1st-round treatments application caused insignificant mortality as compared to the 2nd- and 3rd-round treatments application. It is because of that the scale insects' body is covered with external protective scale layers in which a single spray cannot penetrate/reach the scale insects unless otherwise a redundant spray could be applied. At both experimental sites, the lowest mean percent mortality of *A. tubercularis* was recorded at the first round (10 days after) treatment application, while the highest was recorded after the 3rd round (30the day) of treatment applications.

*Aulacaspis tubercularis* nymphs and adults mean percent mortality due to application NLPWE at Uke Kersa and Arjo Gudetu Farmers Association are shown in **Table 3**. The minimum mean fatality percent was observed in females followed by males and crawlers in that order.


*\* Within a column means go along with alike letter (s) are not notably different from each other (P < 0.05) by Tukey's studentized range test (HSD).*

#### **Table 3.**

*Insecticidal activity of* A. indica *LPWE treatment application on sessile white mango scale nymphs and adults at different stages and sexes due to different concentrations of treatments application at Uke Kersa and Arjo Gudetu farmers' experimental sites.*


**Table 4.**

*LC50 and LC95 of* A. indica *LPWE application against* A. tubercularis *days after treatment (DAT) application at Arjo and Uke Kersa farmers' association administration experimental sites.*

The contact toxicity of *A. indica* LPWE against white mango scale nymphs and adults of *A. tubercularis* is shown in **Table 4**. The LC50 values of *A. indica* LPWE at Arjo Farmers Association administration experimental site against sessile nymphs

*Potency of* Azadirachta indica *L. Leaf Aqueous Extract Insecticide against* Aulacaspis*… DOI: http://dx.doi.org/10.5772/intechopen.110256*

and adults of *A. tubercularis* during 1st, 2nd, and 3rd round treatments application were 15.4, 8.9, and 5.1 mg/100 ml, respectively, while the Chi-square (X<sup>2</sup> ) values with 1st, 2nd, and 3rd round treatments application were 118.0, 155.4, and 212.7, respectively. The LC50 values of aqueous *A. indica* leaf powder at the Uke site against sessile *A. tubercularis* at 1st, 2nd, and 3rd round treatment application were 28.2, 11.4, and 4.90 mg/100 ml, respectively, while the Chi-square (X<sup>2</sup> ) for 1st, 2nd, and 3rd round treatments application were 94.8, 65.6, and 267.5, respectively.

At Arjo Farmers experimental site, the contact toxicity for essential extracts of aqueous *A. indica* LPWE at 1st round treatment application against different stages of *A. tubercularis* adult nymphs, females and males had the LC50 values of 10.6, 24.5 and 14.0 μg/ml, respectively.

The contact toxicity of aqueous *A. indica* leaf powder at 2nd round treatments application against adult nymphs, females, and males had LC50 values of 5.5, 18.3, and 7.9 μg/ml and for the 3rd round treatments application had LC50 values of 3.5, 10.2, and 4.5 μg/ml, respectively. The sessile nymphs and adults of *A. tubercularis* fatality rate count were made consequently 10 days after each round of treatment application. With the same activity at Uke Kersa farmers' association administration experimental site, the essential extracts of *A. indica* LPWE application for 1st round treatments against different stages of *A. tubercularis* adult nymphs, females, and males showed LC50 values of 17.8, 49.7, and 24.7 μg/ml, respectively. The contact toxicity of *A. indica* LPWE for 2nd round treatments application against *A. tubercularis* nymphs, adult females, and males showed LC50 values of 9.9, 17.6, and 11.1 μg/ml, respectively. With the same treatments of *A. indica* LPWE, at 3rd round application against *A. tubercularis* of nymphs, adult females and males showed

#### **Figure 2.**

*Study area map for evaluation of neem (*Azadirachta indica*) leaf powder potency against* Aulacaspis tubercularis *in Oromia regional state, Western Ethiopia.*

#### **Figure 3.**

*The log concentration and probit mortality regression line graph for* A. indica *LPWE treatments against sessile WMS (nymphs, adult females and males) after 1st, 2nd, and 3rd round treatment at Uke Kersa farmers' association administration experimental site.*

LC50 values of 4.8, 6.8, and 4.4 μg/ml, respectively. At both experimental sites, the Chi-square values were significant at a P ≤ 0.05 level at the treatments were promising for the management of sessile *A. tubercularis.*

At Arjo Farmers Association administration treatment site, the log-probit regression line calculated (**Figure 2**) values were Y = 1.5x + 3.2 (X-0.6), Y = 1.0x + 4.0 (X-0.4), and Y = 1.1x + 4.3 (X-0.3) for 1st, 2nd, and 3<sup>d</sup> round treatments application, respectively. Likewise, at Uke experimental site (**Figure 3**) the log-probit regression line calculated values were Y = 1.2x + 3.3 (X-0.5), Y = 1.9x + 2.9 (X-0.8), and Y = 1.7X + 3.9 (X-0.5) for 1st, 2nd, and 3rd round treatments, respectively. At both experimental sites, the R<sup>2</sup> values of the three-round treatment application showed significant differences (**Figures 2** and **3**). The result reviled that the regression model analysis showed notable differences that indicate the mortality caused by the tested three *A. indica* LPWE concentrations showed notably different outputs, designated that the fatality brought about during the 3rd cycle treatment application (30 DAT) showed progressive output than the 1st and 2nd cycle treatment applications. The essential extracts of *A. indica* LPWE application at Arjo and Uke Kersa farmers association administration experimental sites against sessile *A. tubercularis* adult females, males, and crawlers are shown in **Table 5**. The increment in the likelihood of mortality rate was commensurately significant at lower log treatment concentrations than the higher ones, so the regression bar was raised at the lower tips than at the higher tips.

In both study sites, there was statistically significant (p < .01) positive correlation between aqueous *A. indica* LPWE, location, and days after treatment (DAT) with *sex* and mortality of white mango scale populations as depicted in **Table 6**. Moreover, there was a slight negative correlation between both locations (experimental sites)

*Potency of* Azadirachta indica *L. Leaf Aqueous Extract Insecticide against* Aulacaspis*… DOI: http://dx.doi.org/10.5772/intechopen.110256*


*\*AF = Arjo Farmers; UK = Uke Kersa; WMS = white mango scale; DAT = days after treatment; LC50 and LC95 values are expressed as percentage; LC50 = median lethal concentration.*

#### **Table 5.**

*Different concentration of* A. indica *LPWE applications for LC50 and LC95 against* A. tubercularis *nymphs*, *adult females and males (n = 360) 10th, 20th, and 30th DATA application at Arjo farmers and Uke Kersa experimental sites.*


#### **Table 6.**

*Correlations of location,* A. indica *LPWE treatments and days after treatment application (DATA) with sex and mortality of* A. tubercularis *populations in Uke and Arjo farmers orchards.*

and mortality of *A. tubercularis* nymphs, adult females and males with *A. indica* LPWE treatments application, which could be due to slight agro-ecological differences of the study sites with some basic climatic factors such as temperature, relative humidity, and rainfall.

#### **4. Discussion**

The current study illustrated the potency level of *A. indica* LPWE for the management of the white mango scale, *A. tubercularis.* The study by [32] on neem-borne molecules as eco-friendly control tools against mosquito vectors of economic importance revealed that neem *A. indica* extracts are an eco-friendly pest control tool, which has an attractive crown of deep-green foliage available throughout the year. The present study revealed that the application of *A. indica* LPWE is helpful for the management control of sessile *A. tubercularis*. At both study sites, the mortality percentage of *A. tubercularis* with the use of *A. indica* LPWE showed moderately a slightly different mortality result, suggesting that there are some basic climatic factors variations. Nonetheless, there were notable variations observed in mortality between nymphs, adult females, and males of *A. tubercularis* because of variations in their vulnerability to the administration of *A. indica* LPWE. Moreover, adult females' have more tolerance in terms of relatively less mortality than nymphs and male against *A. indica* LPWE at Arjo and Uke Kersa experimental sites, signifying that the growth stages and sex of the insect adversely responded to the application of the *A. indica* LPWE treatments. A study by [33] stated that the mixture of *A. indica* seed oil (Trilogy) against the white mango scale was effectual which caused 76.92 and 81.03% fatality in adults (females and males) and the nymphs, respectively. In contrast, the present study showed significant difference among mortality of male, female, and nymphs at different concentrations of *A. indica* LPWE at both experimental setups. A study by [9] also mentioned that the application of *A. indica* seed powder water extract (SPWE) treatments caused mean mortality of 86 and 83% in sessile *A. tubercularis* at Arjo Gudetu and Uke Kersa experimental sites of east Wollega zone of Oromiya Regional State, western Ethiopia. The high Chi-square values in the treatments probably indicated the heterogeneity of the tested populations. Different concentration levels of *A. indica* LPWE influenced the mortality of sessile *A. tubercularis* (nymphs, adult females, and males) differently. At both experimental sites, the control plot (distilled water) did not show significant mortality rather than natural death.

The outcome of the current study indicated that the *A. indica* LPWE at a 5% concentration against the white mango scale can be utilized as a protective implementation to reduce initial infestation. Furthermore, the result of our study indicated that accurate administration of *A. indica* LPWE including the underside leaf surfaces resulted in an effective management of the white mango scale.

Tolerance of *A. tubercularis* adult females to *A. indica* LPWE treatments could be because of the presence of wax cover or hard external scale coverage (exsuvial) of the insect, which is more impenetrable than the nymphs and males' wax cover. The result of our study goes with the result of [34], which mentioned that the muscular impenetrable wax covering that covers the body of the insect bear a protective obstruction against corporal and insecticide interference.

#### *Potency of* Azadirachta indica *L. Leaf Aqueous Extract Insecticide against* Aulacaspis*… DOI: http://dx.doi.org/10.5772/intechopen.110256*

The finding of the present study also indicated that the botanical formulations from *A. indica* LPWE could put back the use of synthetic insecticides in Integrated Pest Management (IPM) programs. The finding of this study goes in line with the result of [33] who mentioned that neem derivative botanicals have an insecticidal effect for scale insect control and are environmentally friendly and useful in reducing the environmental pollution. He also stated that in the present situation, the neem (*A. indica*) derivative is a vigorous botanical insecticide selected for organic agriculture that is widely used in many countries around the world either singly or in IPM or in coexistence with commercial pesticides. The findings of the present study is supported by [35] results, which mentioned that neem derivative compounds are an eco-friendly botanical insecticide against armord scale insects and mealybugs, and it has a considerable reaction as an insecticide on the population reduction of white mango scale *(Aulacaspis mangiferae).* The study also revealed that *A. indica* crucial oils can be used likely as a substitute source for advancing bio-insecticides development against scale insects.

This showed that the resident community can use *A. indica* LPWE to manage the invasion of *A. tubercularis* in plantation mangoes. Accordingly, the output of this study revealed that the 15% concentrations of *A. indica* LPWE at three or more sequences/frequency of application has a better efficacy on population depletion, and likely it can be utilized for the control of the white mango scale, *A. tubercularis.*

#### **5. Conclusion**

The insecticidal properties of natural plant products have been known since the ancient times. Among the various plant products used as insecticides, the formulations developed from neem (*A. indica*) have shown promising result for insect pest management. The botanical formulations derived from *A. indica* are a nontoxic, biodegradable, and environmentally friendly. In this regard, considering the high risks of chemical insecticides on environmental safety, natural enemies, and animals as well as the human beings, the botanical extract are valuable, cheap, safe, and environmentally friendly alternative insect pest management options. In Ethiopia, considering the management options toward *A. tubercularis* control is almost nil or little, which enabled the pest to invade the whole mango-growing parts of the country. Thus, the result of this study presented an encouraging outcome that *A. indica* aqueous LPWE at 15% application has a promising result to knock down *A. tubercularis* population. Therefore, the aqueous *A. indica* LPWE as a derivative botanical pesticide, besides to implementation of cultural management practices of *A. tubercularis,* such as consistent scouting and periodic inventory systems as well as periodic pruning for *A. tubercularis* infestation, removal, and burning of the infested branches, are important practices as part of IPM.

Thus, the development and expansion of the neem (*A. indica*) tree in mangoproducing belts of the country by incorporating into the "Green Legacy" program of the country could help for eco-friendly and nontoxic actions of pest control.

In addition, research actions on technological advancement in the utilization of neem insecticide should need exceptional consideration in the future plan of action with mango development programs.

#### **Acknowledgements**

We would like to thank HU, WU, and Ethiopian MoE for financial support partly in realizing this research work. Our appreciation also goes to Diga & Guto Gida Districts of Agriculture and Natural Resource Offices for their facilitation in availing experimental sites.

#### **Funding**

Haramaya University (HU), Wollega University (WU), and the Ministry of Education (MoE), made financial support partly for the research grant to the principal author.

#### **Authors' contributions**

Temesgen F. conceptualizes the study design, accomplishes the experiments and statistical analysis, and writes down the document. Prof. Emana G., Dr. Mulatu W., and Prof. Kebede Woldetsdik advise the principal author all over the work and evaluate the document. All coauthors look through and accepted the final document for publishing.

#### **Conflict of interest**

The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest so that the authors declared that there has no conflict of interest.

#### **Data availability statement**

Primary raw data not presented in this study are available as supplementary material on request from the first author without undue reservation.

*Potency of* Azadirachta indica *L. Leaf Aqueous Extract Insecticide against* Aulacaspis*… DOI: http://dx.doi.org/10.5772/intechopen.110256*

#### **Author details**

Temesgen Fita1,2\*, Emana Getu3 , Mulatu Wakgari1 and Kebede Woldetsadike1

1 School of Plant Sciences College of Agriculture and Natural Resources, Haramaya University, Ethiopia

2 Faculty of Agriculture and Natural Resources, Department of Plant Sciences, Wollega University, Ethiopia

3 Department of Zoological Sciences, Addis Ababa University, Ethiopia

\*Address all correspondence to: temefita@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

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### **Chapter 7**

## Perspective Chapter: Current Situation of Insecticide Residues in Food Commodities and Possible Strategies for Management of Residues

*Banka Kanda Kishore Reddy, Addanki Maneesha, Chinna Babu Naik, Malleswari Sadhineni, Tejaswi Yelleti and G. Raja Reddy*

#### **Abstract**

Pesticides have evolved into a crucial instrument in agriculture's evolution as a plant protection agent for increasing food output. Moreover, pesticides contribute significantly by preventing a number of terrible diseases. However, both occupational and environmental pesticide exposure can lead to a number of health issues in people. It has been noted that pesticide exposures are becoming more and more associated with immune system suppression, hormone disruption, lowered intellect, abnormalities in reproduction, and cancer. Because of the great demand for farm produce and their lack of awareness of the hazardous consequences of pesticide residues in food, some farmers do not wait long enough for the residues to wash off after spraying before harvesting. As a result, residues in food products have appeared as a result of increased pesticide use in agriculture. Some of the primary tactics for reducing human exposure to pesticides are pesticide safety, regulation of pesticide usage, appropriate application technology, and integrated pest management.

**Keywords:** pesticide, pesticide residues, environmental pesticide exposure, pesticide safety, integrated pest management

#### **1. Introduction**

The usage of pesticides is common to ensure high agricultural yields. They are employed in the production of agricultural products as well as their post-harvest handling. The growing usage of chemical pesticides, however, has had a negative impact on human health as well as contaminated the environment. It has long been of great concern that food products may contain pesticide residues. When these products are eaten fresh, the issue becomes even more serious. Pesticides have been linked to a variety of risks to human health, from immediate effects like headaches and nausea to long-term ones like cancer, reproductive damage, and endocrine disruption. Pesticides applied during the fruit growth period can dissipate faster because of the growth dilution effect [1]. However, when applied after fruit growth, they are likely to be carried over to the harvested produce and processed products. Processing is considered as effective tool which implies transformation of fresh commodity into valueadded product and ultimately affect the nature and magnitude of residues and during processing pesticide residues may increase or decrease in the transformed product [2]. Processing Factor (PF) is used to assess the risk associated with the consumption of pesticide residues, particularly for processed food products [3].

For pest management and the eradication of disease vectors, developing nations (like China) frequently utilize insecticides (including organophosphorous and pyrethroid) and fungicides (including triazoles and chloronitriles). Pesticide poisonings are far more common in poorer nations than in developed ones as a result of poor pesticide handling procedures, farmers' use of more harmful pesticides, and insufficient monitoring and oversight of these chemicals. Because of a lack of funding and the absence of strict regulations, pesticide residue control initiatives are frequently ineffective in poor nations.

#### **1.1 Insecticide residues found in various food commodities**

Insecticide residues discovered in various food commodities were tabulated in **Table 1**.

#### **1.2 Management strategies to reduce pesticide residues**

#### *1.2.1 Effect of processing on the level of pesticide residues in various fruits and vegetables*

Consumers have little control over pesticide residues that are left in food products in various proportions after harvesting and which are damaging to human health. Hence, pesticide residues present a significant challenge to the international trade in food products. Since pesticide-treated food crops always retain varying amounts of these chemicals, finding non-toxic methods for decontaminating food are essential. The molecular composition, product mix, and environmental conditions all have an impact


#### **Table 1.**

*List of insecticides found in various food commodities.*

*Current Situation of Insecticide Residues in Food Commodities and Possible Strategies… DOI: http://dx.doi.org/10.5772/intechopen.111748*

on the levels of pesticides in various food items. Washing is the most popular technique of processing, and it is an important first step in both home and industrial preparation. As a result, it is critical to consider techniques that may successfully assist in reducing residue content at the individual level in order to limit dietary pesticide exposure.

The effects of commonly used household processes such as washing by tap water, saltwater, lukewarm water, lemon water, tamarind water and ozone water are discussed below.

Tap water washing for 2 minutes eliminated 30–50% of phosalone residues and 65.3% of chlorpyrifos residues [11, 12], whereas tap water washing for 10 minutes removed 53.4, 53.3% dimethoate residues in grapes [13, 14]. Awasthi [15] found that washing mangoes with tap water removed 66–68% of the dimethoate and fenitrothion. Washing guava with tap water reduced dimethoate residues by 42.5–45.9% [16].

Other fruit crops viz., mangoes where 66% dimethoate residues were removed by tap water washing [15], 45.9% in guava [16]. Washing with salt water (2%) solution for 10 min was recorded as an effective decontaminant in removal of acephate, chlorpyrifos, quinalphos, bifenthrin residues (51.80–72.80%), acephate (72.74%), chlorpyrifos (67.52%) and quinalphos (65.0%), respectively in grapes [17–20], imidacloprid (61.89%) in field bean [21, 22], tetraniliprole (61.49%) in tomato [23] and NaCl (5%) removed 90% of quinalphos and profenofos in chili [24].

Pesticide residues combine with sodium chloride solution, a powerful electrolyte solution, which lowers their concentration and offers an appealing source for pesticide residue removal. When dipped in the solution, neonicotinoids with high water solubility were easily separated from the fruits in salt media. Vijayasree et al. [25] discovered that tamarind water (2%) and salt chloride (2%) solutions eliminated 85.56 and 100% of the emamectin benzoate in cowpea pods, respectively. Buprofezin residues in oranges were reduced by 36.50 and 27.51%, respectively, after washing with tap water and salt chloride solution (2%) [26].

Citric acid, the active component of lemon water, was mostly responsible for the residue elimination. The findings supported research in which a 52.2% reduction in dimethoate in tomato [16] was noted. Dimethoate and quinalphos were both eliminated by washing in lemon water (1%) for 10 minutes, along with 45% of the pesticide acetamiprid from tomatoes [27, 28].

The removal of residues by tamarind water solution is due to its acidic nature which is contributed by furan derivatives and carboxylic acids [29]. Studies where tamarind water (2%) washing resulted in a 69.1% reduction of chlorpyriphos in tomatoes, 58.8 and 80.4% reduction of dimethoate and quinalphos in grapes, 58.8% of dimethoate in grapes [14, 16, 27].

It is evident that the ozone concentration administered, the physical characteristics of the food matrix, and the residual ozone present in the medium all affect how effective ozone intervention for pesticide degradation is. The parameters that affect the clearance rate include the application environment (pH, temperature, and humidity), the application method (aqueous vs. gaseous), the ozone concentration, the rate of formation, and the geometry-size of pesticide residue [30]. Dipping apples in ozonated water of 0.25 ppm resulted in reducing the levels of azinophos-methyl on the surface of apples to 75% [31]. Ikeura et al., [32] studied the effect of ozone water (2.0 mg L-1) for 10 min on the level of fenitrothion residues in strawberries and removal rate were concluded as 25%. Removal of chlorpyrifos in lychee fruits with aqueous ozone water concentrations of 2.2, 2.4, 3.2 and 3.4 mg L-1 for 10, 20, 30 and 60 min resulted in 0, 25.8, 29.7 and 67.4% reductions, respectively. Similarly, fumigation of O3 at 80, 160, 200 and 240 mg L-1 for 10, 20, 30 and 60 min resulted in 10, 18,

30, 45% reductions, respectively [33]. Treating the citrus fruits with ozonated water (10 mg L-1) for 5 min reduced the chlorpyrifos by 94.2% [34]. Washing of strawberries in ozonated water (1 mg L-1) for 5 min resulted in removal of chlorpyrifos by 71.5% [35]. Washing of tomatoes in ozonized water for 30 seconds removed chlorpyrifos by 86% [36].

To ensure that customers are not exposed to any health hazards, monitoring pesticide residues ingested through food is necessary. Few foods are consumed without processing, including washing, peeling, drying and pasteurization. During harvest, the residues left on the fruits can be carried into processed foods, such as juice, squash, jams, jelly, and raisins [37]. It is well established that food processing affects residual pesticide concentrations. Therefore, when fruits are processed, it is predicted that the residues will decay due to exposure to various processing procedures. Consequently, it is critical to include processing factors when assessing pesticide residues in processed foods.

Camara et al., [38] conducted various food processing procedures viz., cutting, washing and drying in lettuce to monitor the behavior of imidacloprid and they concluded that PF was 0.53 for imidacloprid which indicates reduction of residue content than in fresh lettuce due to food processing. Pasteurization resulted in the loss of 60.42–100% imidacloprid residues in tomato juice and paste [39, 40]. Pasteurization was found to reduce imidacloprid residues (32.45%) in strawberry juice preparation [40]. Imidacloprid residue reduction (82.66% and 66.55%) in sugared pulp and paste of winter jujube [41], 50.64 and 84.41% removal of imidacloprid residues during strawberry syrup and jam preparation, respectively [40]. Hot air over drying reduced imidacloprid residues by 70% in pomegranate, 36.73% in zucchini processing, 53% in lettuce [38, 42, 43], respectively. Processing of apples were concentrated to 0.162, 1.039, 0.102, 0.049 from 0.061, 0.372, 0.047 and 0.02 mg/kg of quinalphos, chlorpyrifos, cypermethrin and deltamethrin respectively in apple juice than in unprocessed apples [44]. Commercial processing of tomato fruits into tomato juice (under hot break) reduced 100% of imidacloprid residues [28]. Cypermethrin residues were concentrated in seedless variety of grapes to 0.46 ppm when compared to residues in fresh grapes (0.40 ppm), a study conducted by [2]. Producing apple juice from freshly harvested apples resulted in reduction of chlorpyrifos and methomyl residues by 100 and 78.1%, respectively [45].

Reddy et al., [46] studied processing effect on pesticide residues in grapes where Processing factor was calculated and was in the range of 0.01 to 0.35, 0.04 to 0.39 and 0.03 to 0.40 for juice, squash and raisin, respectively. In this study, imidacloprid was removed (59.75–67.94%) from grapes while, washing with water. Washing reduced chlorpyrifos residues (21%) in apple processing [47]. It is inferred that there is a strong correlation between water solubility (600 mg L-1) and removal of imidacloprid [48]. Crushing/homogenization does not impact residues, but it speeds up processes like hydrolysis, which releases isolated enzymes and acids from the cuticle layer more quickly, reducing residues in the juice. Partitioning characteristics of insecticide between pulp and juice are responsible for the low residual levels in juice and squash.

Clarification of juice may eliminate residues retained in the suspended particles. A negligible number of systemic insecticides might be absorbed by pulp or fruits [48]. Studies reported 93.26–97.85% removal of imidacloprid residues during processing of apples into juice [47]. Pesticide residues were significantly reduced during juice processing also reported [44, 45]. Pasteurization was found to reduce 60.42–100% of imidacloprid in tomato juice and paste [28] and imidacloprid residues (32.45%) in strawberry juice preparation [40]. During squash preparation, addition of sugar

#### *Current Situation of Insecticide Residues in Food Commodities and Possible Strategies… DOI: http://dx.doi.org/10.5772/intechopen.111748*

syrup to the juice reduced residues in 94.32% in present study as water was added in sugar syrup resulted in dilution of residues. Hendawi et al., [40] reported imidacloprid residue reduction during strawberry syrup (50.64%) and jam preparation (84.41%) and 66.55 and 82.66% reduction in sugared pulp and paste of winter jujube, respectively [41]. Because of evaporation and degradation, the drying process may have significantly reduced residual levels [49]. With regard to imidacloprid residues, 70.00% reduction in pomegranate by hot oven air drying [42], 53.00% in lettuce [38] and 36.73% reduction in zucchini processing [43], were reported.

In raisin preparation, residues of phosalone (68.04%) and ethion (69.55%) were removed [50]. The processing factor achieved for hexythiazox and bifenazate were 0.36 and 0.15 in grapes for raisin [51]. It is concluded that pesticides with low Kow may be removed through volatilization after drying and this is correlated with studied chemical imidacloprid where the Kow is low (0.57). Catherine et al., [52] reported that water solubility of a pesticide is an important factor during the juicing operation and pesticides with the highest water solubility were present in relatively higher amounts in the juiced carrots, tomatoes and strawberries [53]. Moreover, dimethoate is xylem mobile due to its low log Kow value of 0.7 and phloem mobile due to its pKa of 2. This is probably why washing and peeling are less effective at removing dimethoate than other organophosphates such as chlorpyrifos and parathion and thereby ending up in the filtered juice. Concentration of dimethoate quinalphos, chlorpyrifos, cypermethrin, deltamethrin were found in apple [44], chlorpyrifos in apple juice [47], in wine [54].

Pesticides residue levels were reduced during processing of food commodities but those pesticides (dimethoate, azoxystrobin, pyrimethanil) were not having a preferential partition between liquid and solid phase may be concentrated in the final processed product [55]. The poor transfer/presence of lower residues in filtered juice might be due to low water solubility (0.024 g/L) and high octanol co-efficient (Kow = 5.0) reported for emamectin benzoate, fenpropathrin and propargite in tea brewing [56, 57]. Lower residues of emamectin benzoate in grape and its processed products is might be due to high octanol co-efficient (Kow = 5.0) makes immobile in plant tissues. Our results are in line with studies where post-harvest processing and decoction of Chinese medicinal plant mugua resulted in 99.94% reduction of emamectin benzoate [58] PF of 0.06 in Chinese peony [59]. The processing of grapes into juice, squash and raisin resulted in reducing the residues as well as processing factor less than for imidacloprid, emamectin benzoate and dimethoate [46].

#### **2. Conclusions**

To reduce residue prevalence in food commodities, the governing bodies should undertake pesticide policy awareness campaigns and impose mandatory training to the grape farmers on pesticide usage, the consequences of excessive/improper pesticide use on the environment and the consumers.

#### **Acknowledgements**

The concept was perceived by Chinna Babu Naik and Malleswari Sadhineni. The book chapter was written by BKK Reddy and Maneesha. Book chapter was thoroughly reviewed by Rajareddy and Tejaswi Yelleti.


#### **Appendices and nomenclature**

### **Author details**

Banka Kanda Kishore Reddy1 \*, Addanki Maneesha<sup>2</sup> , Chinna Babu Naik3 , Malleswari Sadhineni1 , Tejaswi Yelleti4 and G. Raja Reddy5

1 Krishi Vigyan Kendra, Acharya NG Ranga Agricultural University, Guntur, India

2 Department of Entomology, Sri Krishnadevaraya College of Agricultural Sciences, Ananthapuramu, Andhra Pradesh, India

3 Indian Institute of Rice Research, Hyderabad, India

4 National Institute of Plant Health Management, Hyderabad, India

5 Indian Agricultural Research Institute, New Delhi, India

\*Address all correspondence to: bankakishorereddy@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Current Situation of Insecticide Residues in Food Commodities and Possible Strategies… DOI: http://dx.doi.org/10.5772/intechopen.111748*

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#### **Chapter 8**

## Determination of Insecticidal and Larvicidal Activities of *Artemisia herba-alba* Essential Oil against *Tribolium confusum*

*Bouzenna Hafsia, Dorsaf Ben Jeddou, Hadil Kadri, Guesmi Fatma, Abassi Raoudha and Najla Hfaiedh*

#### **Abstract**

The present work was to evaluated the insecticidal activity of the essential oil extracted from an aromatic plant *Artemisia herba alba* (Lamiaceae) on larvaes and insects of *Tribolium confusum*. The results obtained showed that the essential oil *A. herba alba* revealed significant antiradical activities against the DPPH radical and a powerful reducing power. The repellent effect and the toxicity by contact were tested at different concentrations (2, 4, 6, 8 μl/ml). The results revealed that the essential oil of *A.herba alba* was moderately toxic after 24 h towards the insects and the larvaes. While the repellent effect showed that *Artemisia oil* had a very strong repellent effect on the larvae's. These results added to those obtained by other authors on the usefulness and effectiveness of essential oils in the control of different.

**Keywords:** *Artemisia herba alba*, essential oils, toxicity, *Tribolium confusum*, repellent effect

#### **1. Introduction**

Plants represent an immense source of complex chemical molecules exploited by man in the perfume, food, cosmetics and pharmaceutical industries. Most plants contain essential oils; they are then called "aromatic plants". These essential oils were found in many parts of the plant: wood, leaves, fruits, bark, seeds and roots. These are complex mixtures made up of several dozen or even more than a hundred compounds, mainly terpenes and aromatic compounds.

In addition, plant production of economic interest often suffered damages caused by pests, disease agents such as viruses, bacteria, insects or fungi. To control the crop enemies, producers had often used synthetic chemicals, including insecticides, fungicides, bactericides and herbicides. Unfortunately, the application of these chemicals had caused unexpected problems both in terms of soil contamination by non-degradable chemical molecules and on human and animal health by the harmful residues that persist on food.

In order to limit these drawbacks, several academic and public laboratories had taken an interest in the development of biological control [1]. In this work, was interested in a medicinal plant, it is *Artemisia herba-alba*, for which, it's essential oils was extracted, and evaluated its possible interest in the context of biological control.

*A. herba-alba* has been used in traditional medicine by many cultures since ancient times. It is a very popular remedy that is often used to facilitate digestion, calm abdominal pain and certain liver ailments. Its roots were indicated against certain nervous disorders [2]. Their aqueous extract were traditionally used in Jordan as an antidote against the venoms of several types of snakes and scorpions. In North Africa, it was known to treat bronchitis, abscess, diarrhea and as a vermifuge. Its oil essential had many activities including antioxidant, hypoglycemic, antifungal, antibacterial [3, 4].

In this context, the study was evaluated the bioinsecticide effect of the essential oil of *A. herba-alba* on adults and larvae of *T. confusum*.

#### **2. Materials and methods**

#### **2.1 Extraction of essential oil from** *A. herba alba*

#### *2.1.1 Plant material*

The biomass used for the extraction of essential oils was composed of the aerial parts of *A. herba alba*. They were collected from Jbel Mdhila mountain of the Gafsa region in April 2022. The aerial parts of this plant were dried in the open air, away from light, for 8 days. After drying, the dried plant material was ground in a mortar to increase the solvent-sample contact surface.

#### *2.1.2 Chemicals*

Acetone, DPPH, BHT, potassium ferricyanide, trichloroacetic acid were purchased from Sigma-Aldrich Chemicals Co. (St. Louis, MO, USA).

#### **2.2 Extraction of volatile oils**

The extraction of essential oils was done by the method of hydrodistillation: Hydro-distillation is a technique for extracting essential oils that is done using a simple distillation. In a 1 liter flask, 500 mL of distilled water was added with a few pumice stones to regulate the heating and about 50 g of the dried leaves of plant. The water was brought to a boil, taking care not to heat until completely dry. Thus, the water vapor entrains the volatile organic substances which condense in the refrigerator then flow drop by drop and are collected at the other end of the assembly in the graduated cylinder. The volume of the distillate is approximately 130 mL.

#### **2.3 Determination of extraction yield**

The yield of essential oils is defined as being the ratio between the mass of the essential oil obtained and the mass of the plant material treated.

*Determination of Insecticidal and Larvicidal Activities of* Artemisia herba-alba *Essential Oil… DOI: http://dx.doi.org/10.5772/intechopen.110623*

$$\mathbf{R} = \left(\frac{\mathbf{m}}{\mathbf{m}\mathbf{0}}\right) \times \mathbf{1}0\mathbf{0} \tag{1}$$

With R: essential oil yield (%), m: mass in grams of the essential oil, m0: mass in grams of dry plant matter.

#### **2.4 Study of biological activities**

#### *2.4.1 Antioxidant activity of essential oil.*

The antioxidant activity was tested using two methods: scavenging of the free radical DPPH and determination of its reducing power (FRAP).

#### *2.4.2 The DPPH• (2,2-diphenyl-1-picrylhydrazyl) test*

DPPH• (2,2-Diphenyl-1-picrylhydrazyl) is a stable purplish-colored free radical that absorbs at 517 nm. In the presence of anti-radical compounds, the DPPH• radical is reduced and changes color by turning yellow. This method is based on measuring the capacity of antioxidants to scavenge the 2,2-diphenyl-1-picrylhydrazil (DPPH•) radical. The latter is reduced to the form of hydrazine (non-radical) by accepting a hydrogen atom. The effect of oils on DPPH is measured by the procedure described by Öztürk et al. [5]. The percentage of DPPH anti-radical activity is calculated according to the equation:

#### *2.4.3 Reducing power (FRAP)*

The reducing power of ferric ions (Fe3+) of an extract was evaluated using the method described by Oyaizu [6]. A volume of 1.25 mL of a phosphate buffer solution (0.1 M, pH 6.6) and 1.25 mL of potassium ferricyanide (1% w/v) were mixed with 500 μL of an extract solution at concentrations variables. The reaction mixture was incubated for 30 minutes at 50°C, then a volume of 1.25 mL of 10% w/v trichloroacetic acid was added to stop the reaction, the mixture was centrifuged at 1500 × g for 10 min. A volume of 1.25 mL of the supernatant is mixed with 1.25 mL of distilled water and 250 μL of a 0.1% w/v solution of FeCl3 then incubated for 10 minutes. The absorbance of the mixture was measured at 700 nm. The greater the absorbance of the reaction mixture, the greater the reducing power. BHT was used as a positive control with the same concentrations (50–300 μg/mL) and under the same experimental conditions was used as a standard antioxidant and the results are presented as the change in absorbance with concentration. From the graph the effective concentration (EC50) is determined which gives an absorbance of 0.5. The lower the EC50, the stronger the reducing power. The test measuring the reducing power was repeated in three trials for each extract.

#### **2.5 Study of the insecticidal and larvicidal activity of the essential oil of**  *A. herba alba*

The toxicity tests of *A. herba-alba* on insects and larvae of *T. confusum* were carried out according to two modes of penetration, one penetration by contact and the other by repellent effect.

#### **2.6 Evaluation of the toxicity of this oil by contact effect**

After preparing the doses, each solution was spread evenly on a disc of Wattmantype filter paper previously placed in Petri dishes of the same diameter. After evaporation of the dilution solvent, all the boxes are infested by 10 insects or larvae of *T. confusum*. We carried out three repetitions for the four doses of essential oil tested, and the same for the control not treated with the essential oil. A count of the dead insects is carried out after 24 hours of this treatment.

#### **2.7 Test to evaluate the repellent effect of essential oil on insects and larvaes**

The repellent effect of essential oils against adults and larvae of *T. confusum* was evaluated using the technique described by McDonald et al. [7] using the preferential area method on filter paper. In this experiment, the 9 cm diameter filter paper discs used for this purpose were divided into two equal parts. Four different essential oil contents were used in this test: 2, 4, 6, 8 μl/ml [8]. 0.5 mL of each dose was placed on one half of the disc, while the other half received only acetone. The two discs were placed in the open air for 10 minutes, in order to evaporate the acetone. After acetone drying. The filter paper disc was placed in a 9 cm diameter Petri dish. A batch of 10 individuals of *T. confusum* were placed in the center of filter paper. The boxes are placed under ambient temperature conditions to calculate the percentage of repellent value for each essential oil. The percentage of repulsion is thus calculated according to the following formula:

$$\text{Percent Repulsion} \left(PR\% \right) = \left( \left( \text{NC} - \text{NT} \right) / \text{NC} \right) \times 100 \tag{2}$$

Where: NC: number of the individual present on the part of the disc treated only with acetone NT: number of the individual present on the part of the disc treated with the prepared dose. The average percentage repellency for each oil is calculated (PR) and assigned to one of the different repellent classes varying from 0 to V [7] which showed in **Table 1**.

#### **2.8 Data analysis method**

#### *2.8.1 Mortality correction by Abbott's method*

The effectiveness of this essential oil is evaluated by mortality. The results of the tests carried out do not only represent the mortality caused by the oil but there is also the natural mortality. This mortality is corrected using the Abbott formula.

$$\text{MC} \left( \text{\%} \right) = \left( \left( MT - Mt \right) / \left( \mathbf{100} - Mt \right) \right) \times \mathbf{100} \tag{3}$$

\_ MC %: percentage of corrected mortality.

\_ Mt.: percentage of mortality obtained in the control population.

\_ MT: percentage of mortality obtained in the treated population.

*Determination of Insecticidal and Larvicidal Activities of* Artemisia herba-alba *Essential Oil… DOI: http://dx.doi.org/10.5772/intechopen.110623*


#### **Table 1.**

*Percentage of repulsion (PR) according to the classification of Donald et al. [7].*

#### **3. Results and discussion**

#### **3.1 Aspect and yield of the essential oil**

The extraction by hydrodistillation of the aerial parts *of A. herba-alba*, obtained the essential oil of yellow color and very strong smell. The yield of the essential oil obtained in relation to the total weight of the dry plant is 0.16%. The yield was low compared to that obtained by the same plant in different regions of Algeria (from 0.2 to 0.95%) [2, 9]. That of Spain was 0.41 to 2.30% [10] and that of Jordan 1.3% [11]. This difference in yields can be attributed not only to the geographical origin of the plant, but also to many factors such as: growth stage, pedoclimatic conditions, place of production, etc. Several studies had shown the influence of the vegetative cycle and the extraction technique on the yield and quality of EO.

#### **3.2 Evaluation of antioxidant activity**

In order to evaluate the antioxidant activity of *A. herba alba* essential oil, different antioxidant tests (DPPH and FRAP) were used.

#### *3.2.1 Anti-radical activity against DPPH radical*

The antioxidant effect was assessed by the DPPH test as the ability of DPPH •, compounds with antiradical activity, to scavenge free radicals. From these results, it deduced that the evolution of the anti-free radical activity is studied in vitro against the radical DPPH is dose-dependent (**Figure 1**). Indeed, the increase in the concentration of the oil is followed by an increase in the anti-DPPH activity. These results were also expressed by IC50. Indeed, the IC50 parameter is defined as being the concentration of the substrate which leads to a loss of 50% of the activity. Indeed, the oil with the lowest IC50 value exerts the most powerful antiradical activity. Indeed, the oil reported a value of IC50 = 160 μg/mL. This reflected a powerful antioxidant power by comparing it with the standard (ascorbic acid) which has the IC50 value = 210 μg/mL.

#### *3.2.2 Iron reduction test (FRAP)*

The reducing power was based on the reduction of the ferric ion (Fe3+) to ferrous ion (Fe2+) by transfer of an electron or donation of a hydrogen atom.

#### **Figure 1.**

*Antiradical activity of the essential oil of Artemisia herba alba. Each value represents the mean ± SD (n = 3 trials for each sample). HE: Essential oil, Ac: ascorbic acid.*

#### **Figure 2.**

*Graphic representation of the reducing power of* A. herba alba *and BHT each value represents the mean ± SD (n = 3 trials for each sample).*

The **Figure 2** represented the reducing power of essential oil of *A. herba alba* and the BHT standard. The results showed that the reducing power increased with the concentration. The absorbance at 700 nm of the oil increased proportionally to its concentration. However, in comparison with BHT (used as a control), the reducing power of this oil was lower. Indeed, even at high concentration (300 μg/mL), we observed an absorbance of 0.32 for oil against 1.00 for BHT.

#### *3.2.3 Insecticidal and larvicidal activity of the essential oil of A. herba alba*

To evaluate the insecticidal and larvicidal effect of the essential oil of *A. herba alba* the mortality rate of adults and larvae's by contact and repellent method were estimated.

*Determination of Insecticidal and Larvicidal Activities of* Artemisia herba-alba *Essential Oil… DOI: http://dx.doi.org/10.5772/intechopen.110623*

#### **Figure 3.**

*Corrected mortality percentage of T. confusum insects after 24 h of exposure to* A.herba alba *essential oil. Each value represents the mean ± SD (n = 3 replicates with 10 insects each).*

#### *3.2.3.1 Assessment of adult and larval mortality by contact effect*

The corrected mortalities were mentioned in **Figures 3** and **4**. The results indicated that the larvae were more resistant than the insects. The results showed an increase in the concentration-corrected mortality rate. As an aromatic plant, the volatility of the essential oil due to the montepenes and the major Camphor compound of this oil gives it an effectiveness in controlling stock insect pests. These results confirm other inferences reporting Chaied et al. [12] that this oil showed good insecticidal activity.

#### *3.2.3.2 Determination of the repellent activity of the essential oil*

The results of the evaluation of the repellent effects of essential oil of *A. herba alba* on the larvae and adults of *T. confusum* were presented in **Tables 2** and **3**. The percentage of repellent essential oil used increases according to the dose and time.

#### **Figure 4.**

*Percentage of corrected mortality of larvae of* T. confusum *after 24 h of exposure to* A.herba alba *essential oil. Each value represents the mean ± SD (n == 3 replicates with 10 insects each).*


#### **Table 2.**

*Repellent effects on* T. confusum *larvae exposed to* A. herba alba *essential oil.*


**Table 3.**

*Repellent effects on adults of* Tribolium confusum *exposed to the essential oil of* A. herba alba*.*

Our deductions revealed that the highest dose of *A. herba alba* had a strong repellent effect of 100% on *T. confusum* larvaes and adults respectively. From these results, it was concluded that higher concentration of essential oil resulted in maximum pest repellency compared to lower concentrations. According to the classification established by Mc. Donald et al. [7], it can be concluded that the oil recorded a very strong repellent effect against adults and larvae of *T. confusum*. Many researchers have pointed out that certain plant essential oils exhibited strong repellent effects against harmful storage [13–15].

#### **4. Conclusion**

The use of bio-insecticides was a major and effective potential for crop protection. The use of essential oils extracted from aromatic plants has demonstrated their insecticidal activity against insect pests of stocks. In this work, initially, the evaluation of the antioxidant activity by the DPPH free radical scavenging method and the FRAP test showed that the essential oil has a significant antioxidant power. On the other hand, treatment with *A. herba alba* essential oil on *T. confusum* larvae and insects gave a repellent effect and a toxic effect. At the end of this study, the results obtained are in

*Determination of Insecticidal and Larvicidal Activities of* Artemisia herba-alba *Essential Oil… DOI: http://dx.doi.org/10.5772/intechopen.110623*

agreement with previous results. They largely confirm the usefulness of essential oils in the pest control program, particularly *in P. interpunctella* and *E. kuheniella* and in different orders of insects.

#### **Funding**

This research was supported and funded by the Ministry of Higher Education and Scientific Research (MHESR, Tunisia).

#### **Conflict of interest**

The authors declare that they have no conflict of interest.

### **Author details**

Bouzenna Hafsia\*, Dorsaf Ben Jeddou, Hadil Kadri, Guesmi Fatma, Abassi Raoudha and Najla Hfaiedh Laboratory of Biotechnology and Biomonitoring of the Environment and Oasis Ecosystems (LBBEEO), Faculty of Sciences of Gafsa, University of Gafsa, Tunisia

\*Address all correspondence to: bouzenna.hafsia@hotmail.fr

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### *Edited by Habib Ali, Adnan Noor Shah, Muhammad Bilal Tahir, Sajid Fiaz and Basharat Ali*

*Insecticides - Advances in Insect Control and Sustainable Pest Management* offers an in-depth exploration of insect control, showcasing the latest scientific advancements, practical applications, and sustainable solutions. Insects play a crucial role in our ecosystem, but their presence can also present significant challenges to agriculture, public health, and the environment. This book serves as a comprehensive guide to understanding the cutting-edge approaches to insect control, providing valuable insights into the development and implementation of innovative insecticides. Authored by a team of renowned experts, the book delves into the fascinating world of insect biology, examining the intricate mechanisms that drive their behavior and evolution. With a strong focus on sustainable pest management, the book emphasizes environmentally friendly methods that minimize the impact on non-target organisms and reduce chemical residues in the environment. Within the pages of this book, readers will discover a wealth of information on emerging insecticide technologies, including novel chemical compounds, biological agents, and genetic engineering approaches. The effectiveness, safety profiles, and modes of action of these advancements are thoroughly analyzed, equipping professionals and researchers with the necessary knowledge to make informed decisions and develop integrated pest management strategies. Whether you are a scientist, student, or practitioner in the fields of entomology, agriculture, or public health, *Insecticides - Advances in Insect Control and Sustainable Pest Management* is an essential resource that provides a comprehensive understanding of insect control. It addresses critical issues such as insect resistance, regulatory frameworks, and the social and economic implications of insecticide use, paving the way for sustainable pest management practices. Embark on a journey through the intricate world of insects and explore the innovative solutions designed to control them. With up-to-date research, practical applications, and a focus on sustainability, this book is an invaluable companion for navigating the complex realm of insect control in the 21st century.

Published in London, UK © 2023 IntechOpen © Tomasz Klejdysz / iStock

Insecticides - Advances in Insect Control and Sustainable Pest Management

Insecticides

Advances in Insect Control and Sustainable

Pest Management

*Edited by Habib Ali, Adnan Noor Shah, Muhammad Bilal Tahir, Sajid Fiaz* 

*and Basharat Ali*