Bt Research 2024, Vol.15 http://microbescipublisher.com/index.php.bt © 2024 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher.
Bt Research 2024, Vol.15 http://microbescipublisher.com/index.php.bt © 2024 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. MicroSci Publisher is an international Open Access publisher specializing in microbiology, bacteriology, mycology, molecular and cellular biology and virology registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher MicroSci Publisher Editedby Editorial Team of Bt Research Email: edit@bt.microbescipublisher.com Website: http://microbescipublisher.com/index.php/bt Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Bt Research (ISSN 1925-1939) is an open access, peer reviewed journal published online by MicroSciPublisher. The journal is publishing high quality original research on all aspects of Bacillus thuringiensis and their toxins affecting the living organisms, as well as environmental risk and public policy relevant to Bt modified organisms. Topics include (but are not limited to) Bt strain identification, novel Bt toxin discovery and bioassay, transgenic Bt plants, insecticidal mechanism of Bt toxin as well as resistant mechanisms of target-insect to Bt toxin. All the articles published in Bt Research are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MicroSciPublisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.
Bt Research (online), 2024, Vol. 15 ISSN 1925-1939 http://microbescipublisher.com/index.php/bt © 2024 MicroSci Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Latest Content 2024, Vol.15, No.5 【Review Article】 Functional Genomics of Bt: Unveiling Key Genes for Insecticidal Activity 215-222 Xiaoqing Tang DOI: 10.5376/bt.2024.15.0021 Public Health Implications of Bt-based Mosquito Control Programs 223-231 Minsheng Lin DOI: 10.5376/bt.2024.15.0022 【Research Insight】 Synergistic Effects of Bt Toxins with other Insecticidal Proteins 232-239 Hongpeng Wang, Minghua Li DOI: 10.5376/bt.2024.15.0023 【Research Perspective】 Artificial Intelligence in Predicting Bt Toxin-Insect Interactions 240-247 JiaXuan DOI: 10.5376/bt.2024.15.0024 【Feature Review】 Formulation Strategies for Bt-based Biopesticides 248-256 Xian Zhang, Shujuan Wang DOI: 10.5376/bt.2024.15.0025
Bt Research 2024, Vol.15, No.5, 215-222 http://microbescipublisher.com/index.php/bt 215 Review Article Open Access Functional Genomics of Bt: Unveiling Key Genes for Insecticidal Activity Xiaoqing Tang Hainan Institute of Biotechnology, Haikou, 570206, Hainan, China Corresponding email: xiaoqing.tang@hitar.org Bt Research, 2024, Vol.15, No.5 doi: 10.5376/bt.2024.15.0021 Received: 15 Jul., 2024 Accepted: 26 Aug., 2024 Published: 08 Sep., 2024 Copyright © 2024 Tang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Tang X.Q., 2024, Functional genomics of Bt: unveiling key genes for insecticidal activity, Bt Research, 15(5): 215-222 (doi: 10.5376/bt.2024.15.0021) Abstract This study reviewed and analyzed the functional genomics of Bacillus thuringiensis (Bt), focusing on identifying and describing the key genes responsible for its insect virulence. Given the use of Bt products in providing eco-friendly pest management solutions, it is important to have a deep understanding of these genes. Through the latest advances in proteomics, genomics, and bioengineering, we explore the specificity of Bt toxin and its effective mechanism against various pests, and also evaluate the behavior of Bt protein in the environment and its potential impact on microbial ecology, providing new insights into the biosafety and sustainability of biosecurity-based pest control strategies. The research method combined gene expression analysis in the laboratory and data from field experiments to evaluate the function and regulatory network of toxin genes in Bt strains using comparative genomics and multi-omics methods. Studies have shown that environmental factors and the microbial composition of the pest gut can significantly affect the potency of these toxins. Keywords Functional genomics; Bacillus pasteurus; Insect toxicity; Proteomics; Environmental biosecurity 1 Introduction Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming bacterium that has garnered significant attention in agricultural biotechnology due to its potent insecticidal properties. Bt produces parasporal crystals during sporulation, which contain δ-endotoxins (Cry and Cyt proteins) that are toxic to a wide range of insect pests, including those from the orders Lepidoptera, Coleoptera, and Diptera (Caballero et al., 2020; Hang et al., 2021). These proteins are highly specific to their target pests, making Bt-based products an environmentally friendly alternative to broad-spectrum synthetic insecticides. The specificity and biosafety of Bt have led to its extensive use in both Bt transgenic crops and Bt biopesticides, contributing to sustainable pest management practices (Gupta et al., 2021; Li et al., 2022). The use of Bt as a biological insecticide dates back to the early 20th century when it was first discovered and identified for its insecticidal properties. Over the decades, Bt has been developed into various commercial formulations, including sprays and dusts, and more recently, genetically engineered crops expressing Bt toxins. These advancements have significantly reduced the reliance on chemical pesticides, thereby mitigating environmental pollution and the development of pest resistance (Cao et al., 2020). The historical success of Bt-based products is attributed to their ability to target specific pests without harming non-target organisms, including beneficial insects and humans (Kouadio et al., 2021). This study focuses on the identification and characterization of key genes responsible for its insecticidal activity. By studying the latest advances in proteomics, genomics, and bioengineering, the molecular mechanisms underlying the specificity and effectiveness of Bt against various pests are elucidated. In addition, the environmental behavior of Bt protein and its impact on microbial ecology will also be explored, providing insights into the biosafety and sustainability of Bt based pest control strategies, with the hope of strengthening their future research directions in agriculture. 2 Bt Toxins and Insecticidal Genes 2.1 Classification and structure of Bt toxins Bacillus thuringiensis (Bt) produces a variety of Cry and Cyt toxins, which are classified based on their amino acid sequences and insecticidal activity. Cry toxins, such as Cry1, Cry2, Cry4, and Cry9, are known for their
Bt Research 2024, Vol.15, No.5, 215-222 http://microbescipublisher.com/index.php/bt 216 specificity towards different insect orders, including Lepidoptera, Diptera, and Coleoptera (Benfarhat-Touzri et al., 2019; Li et al., 2020; Arsov et al., 2023). These toxins typically form parasporal crystals during sporulation, which dissolve in the insect midgut, releasing the active toxin. The structure of Cry toxins generally includes three domains: a receptor-binding domain, a pore-forming domain, and a protease-resistant core (Liu et al., 2022). Cyt toxins, on the other hand, have a different structure and mode of action, often working synergistically with Cry toxins to enhance insecticidal activity (Valtierra-de-Luis et al., 2020). 2.2 Genomic identification of toxin genes The identification of Bt toxin genes has been greatly facilitated by genomic sequencing and PCR-based screening. For instance, the genome of Bt strain BTG was sequenced, revealing the presence of multiple cry genes, including cry1Ab35, cry1Db, cry1Fb, cry1Ib, cry2Ab, cry8Ea1, and cry9Ba, which contribute to its broad-spectrum insecticidal activity. Similarly, the cry1D-250 gene was identified and cloned from a Bt strain with high toxicity against Spodoptera littoralis, highlighting the importance of genomic tools in discovering new insecticidal proteins (Benfarhat-Touzri et al., 2019). These genomic approaches not only help in identifying the presence of toxin genes but also in understanding their distribution and potential for pest control. 2.3 Functional insights intoCry andCyt genes Functional studies have provided significant insights into the roles of Cry and Cyt genes in insecticidal activity. For example, the Cry2A toxin's interaction with the ATP-binding cassette subfamily A member 2 (ABCA2) receptor in Bombyx mori has been elucidated, showing that mutations in ABCA2 confer resistance specifically to Cry2A without affecting susceptibility to other Cry toxins. Additionally, the Cry1Ac toxin's binding to the cadherin receptor BT-R1 in Manduca sexta triggers a signaling cascade leading to insect death, demonstrating the critical role of receptor interactions in Cry toxin function (Chen et al., 2021). The synergistic activity between Cry10Aa and Cyt2Ba toxins against Aedes aegypti larvae underscores the potential of combining different Bt toxins to enhance insecticidal efficacy. These functional insights are crucial for developing new strategies to manage insect resistance and improve the effectiveness of Bt-based biopesticides. 3 Mechanisms of Insecticidal Activity 3.1 Bt toxin interaction with insect midgut receptors The interaction between Bacillus thuringiensis (Bt) toxins and insect midgut receptors is a critical step in the insecticidal process. The Cry1A toxins, including Cry1Aa, Cry1Ab, and Cry1Ac, bind tightly to the cadherin-like receptor BT-R1 in the midgut of the moth Manduca sexta, initiating a signaling cascade that leads to insect death (Liu et al., 2022). This binding is highly specific, with the toxins competing for the same binding site on BT-R1, localized in the 12th cadherin repeat. The C-terminal region of Cry1Ab protoxin has been shown to provide additional binding sites for alkaline phosphatase (ALP) and aminopeptidase N (APN) receptors, enhancing the toxin's binding affinity and toxicity (Peña-Cardeña et al., 2018). The ATP-binding cassette (ABC) transporters, such as ABCC2 and ABCC3, also play a significant role as receptors for Cry toxins, with their expression regulated by the transcription factor FOXA, which modulates insect susceptibility to Cry1Ac toxin (Li et al., 2017). 3.2 Mode of action of different Bt toxins Different Bt toxins exhibit distinct modes of action, which are influenced by their structural domains and receptor interactions. For instance, the Cry4Ba toxin, specific to mosquito larvae, utilizes its C-terminal domain to interact with the membrane-bound alkaline phosphatase receptor in Aedes aegypti, with specific residues like Leu615 playing a crucial role in this interaction (Thammasittirong et al., 2021). The Cry1Ac toxin's mode of action involves binding to multiple receptors, including cadherin and ABC transporters, with synergistic effects observed when both receptor types are present (Chen et al., 2015). Furthermore, chimeric Bt proteins like Cry1A.2 and Cry1B.2 have been engineered to combine domains from different toxins, broadening their insecticidal spectrum and minimizing receptor overlap, thus enhancing their effectiveness against various lepidopteran pests.
Bt Research 2024, Vol.15, No.5, 215-222 http://microbescipublisher.com/index.php/bt 217 3.3 Role of gut microbiota in Bt toxin efficiency The efficiency of Bt toxins can be significantly influenced by the gut microbiota of the target insects. Studies have shown that the presence of specific gut bacteria can enhance the insecticidal activity of Bt toxins. The midgut histochemistry of Tuta absoluta populations with different susceptibility levels to Bt formulations revealed that the gut microbiota plays a role in the insect's response to the toxin. The presence of certain glycosylation patterns, as indicated by lectin labeling, correlates with higher susceptibility to Bt toxins, suggesting that gut microbiota may modulate the expression or availability of toxin receptors (Oliveira et al., 2018). This interaction between gut microbiota and Bt toxins highlights the complexity of the insecticidal process and the potential for leveraging microbiota to enhance Bt toxin efficacy. 4 Genetic Regulation of Bt Toxin Production 4.1 Key regulatory genes involved in toxin synthesis The production of Bacillus thuringiensis (Bt) toxins is a complex process regulated by various genetic and environmental factors. Several key regulatory genes have been identified that play significant roles in the synthesis of Bt toxins. For instance, the Forkhead box protein A (FOXA) transcription factor has been shown to up-regulate the expression of Cry1Ac toxin receptors ABCC2 and ABCC3 in Helicoverpa armigera and Spodoptera litura, thereby enhancing susceptibility to the toxin. Similarly, the GATAe transcription factor in Helicoverpa armigera has been found to increase the transcription of multiple Cry1Ac receptor genes, including cadherin (CAD), ABCC2, and alkaline phosphatase (ALP), across various insect cell lines, thereby inducing toxin susceptibility (Wei et al., 2019). These findings highlight the critical role of transcription factors in regulating the expression of genes essential for Bt toxin activity. 4.2 Environmental and host factors influencing gene expression Environmental and host factors also significantly influence the expression of Bt toxin-related genes. For example, exposure to sub-lethal doses of Cry1AcF toxin in Galleria mellonella larvae led to the upregulation of several Cry receptor genes, including ABC transporter, alkaline phosphatase, aminopeptidase N, and cadherin, in the midgut tissue (Dutta et al., 2022). Additionally, the MAPK signaling pathway has been implicated in the regulation of midgut ALPand ABCCgenes, which are associated with resistance to Cry1Ac toxin in Plutella xylostella (Guo et al., 2015). These studies suggest that both external environmental conditions and internal host regulatory mechanisms can modulate the expression of genes involved in Bt toxin susceptibility. 4.3 Post-Transcriptional Modifications and Toxin Efficiency 4.3.1 mRNA stability and toxin production The stability of mRNA transcripts encoding Bt toxin receptors is vital for sustained toxin production. For instance, the differential expression of ABCC2 and ABCC3 genes, regulated by transcription factors like FOXA and GATAe, can influence the stability and availability of mRNA for translation, thereby affecting the overall production of toxin receptors. 4.3.2 Translational regulation and toxin protein synthesis Translational regulation is another critical factor in Bt toxin production. The presence of specific regulatory elements in the mRNA can influence the efficiency of translation and the synthesis of toxin proteins (Feng, 2024). For example, the expression of Cry1Ac and Cry3A proteins in transgenic Populus × euramericana 'Neva' plants demonstrated differential protein levels, with Cry3A being expressed at much higher levels than Cry1Ac, indicating the role of translational regulation in determining protein abundance (Ren et al., 2021). 4.3.3 Folding and processing of toxin proteins The proper folding and processing of Bt toxin proteins are essential for their insecticidal activity. Proteomic analyses have revealed that the parasporal crystals of Bt strains, such as GR007, are composed of multiple Cry proteins, each requiring specific folding and processing mechanisms to achieve their active forms (Figure 1) (Pacheco et al., 2021). Additionally, the involvement of proteases like trypsin in the activation of Vip3Aa protoxin in Spodoptera litura larvae underscores the importance of post-translational modifications in the functional activation of Bt toxins (Song et al., 2016).
Bt Research 2024, Vol.15, No.5, 215-222 http://microbescipublisher.com/index.php/bt 218 Figure 1 Genome of B. thuringiensis strain GR007 (Adopted from Pacheco et al., 2021) 5 Mechanisms of Resistance to Bt 5.1 Molecular mechanisms of insect resistance Insect resistance to Bacillus thuringiensis (Bt) toxins is primarily driven by genetic mutations that alter the structure and function of midgut receptors, which are crucial for toxin binding. For instance, mutations in the ABC transporter subfamily C genes, specifically ABCC2 and ABCC3, have been linked to high levels of resistance in several lepidopteran species, including the diamondback moth, Plutella xylostella. CRISPR/Cas9-mediated knockout studies have demonstrated that these genes are essential for the binding of Cry1Ac toxins to midgut brush border membrane vesicles, thereby confirming their role as functional receptors for Bt toxins (Guo et al., 2019). Additionally, other studies have identified mutations in genes coding for surrogate receptors as significant contributors to resistance (Pinos et al., 2021). 5.2 Genomic studies on resistance evolution Genomic approaches have been instrumental in uncovering the mutations responsible for Bt resistance. Research on the European corn borer, Ostrinia nubilalis, has provided significant insights into the genetic basis of resistance. These studies have identified specific mutations that alter the binding receptor structure in the midgut, which directly impacts the efficacy of Bt toxins (Xuan, 2024). Such genomic studies are crucial for understanding the evolution of resistance and for developing strategies to mitigate it (Coates, 2016). Furthermore, comprehensive reviews of field-evolved resistance have highlighted the importance of key resistance genes in maintaining the sustainability of Bt technology.
Bt Research 2024, Vol.15, No.5, 215-222 http://microbescipublisher.com/index.php/bt 219 5.3 Strategies to overcome resistance To counteract the evolution of resistance, several strategies have been proposed. One approach involves the use of multiple Bt toxins with different modes of action to reduce the likelihood of resistance development. Another strategy is the implementation of refuges, where non-Bt crops are planted alongside Bt crops to maintain a population of susceptible insects. Ongoing monitoring and genomic studies can help in the early detection of resistance, allowing for timely management interventions. The use of CRISPR/Cas9 technology to study and potentially modify resistance genes also holds promise for overcoming resistance. 5.4 Case studies on resistance development Case studies on resistance development provide valuable insights into the practical challenges of managing Bt resistance. For example, the diamondback moth, Plutella xylostella, has developed high levels of resistance to Cry1Ac toxins through mutations in the ABCC2 and ABCC3 genes. These mutations result in truncated proteins that fail to bind the toxin effectively, leading to resistance (Figure 2). Another notable case is the European corn borer, Ostrinia nubilalis, where field and laboratory studies have documented resistance evolution due to mutations affecting midgut receptor binding (Jurat-Fuentes et al., 2021). These case studies underscore the need for integrated pest management strategies to sustain the effectiveness of Bt crops. Figure 2 Cry1Ac immunodetection. (A) Larvae exposed to a high dose of Cry1Ac (7 µg) for 20 min. The green fluorescence shows Cry1Ac trapped in the peritrophic matrix (PM) and bound to the brush border membrane (BBM). (B) Control larvae not exposed to the toxin. An Alexa Fluor 488-anti-rabbit antibody was used as secondary antibody (Adopted from Pinos et al., 2021) 6 Genomic Techniques for Bt Strain Improvement 6.1 CRISPR-Cas9 and gene editing approaches CRISPR-Cas9 has emerged as a revolutionary tool in the field of genome editing, offering unprecedented precision and efficiency in modifying genetic material. This technology utilizes a guide RNA (gRNA) to direct the Cas9 endonuclease to specific DNA sequences, enabling targeted gene knockouts, insertions, and modifications. The versatility of CRISPR-Cas9 has been demonstrated across various organisms, including plants and insects, making it a powerful tool for Bt strain improvement. For instance, CRISPR-Cas9 has been successfully employed to enhance crop resistance to diseases and environmental stresses, which can be analogously applied to optimize Bt strains for better insecticidal activity (Arora and Narula, 2017). Despite its potential, challenges such as off-target effects and delivery methods need to be addressed to fully harness the capabilities of CRISPR-Cas9 in Bt strain improvement (Manghwar et al., 2020). 6.2 Comparative genomics for identifying novel genes Comparative genomics involves the analysis of genetic material from different organisms to identify genes that are unique or highly conserved. This approach can be instrumental in uncovering novel genes responsible for the insecticidal properties of Bt strains. By comparing the genomes of highly effective Bt strains with those of less effective ones, researchers can pinpoint specific genes that contribute to enhanced insecticidal activity. These identified genes can then be targeted for further study and manipulation using advanced genomic techniques like
Bt Research 2024, Vol.15, No.5, 215-222 http://microbescipublisher.com/index.php/bt 220 CRISPR-Cas9. Comparative genomics thus provides a foundational understanding that can guide targeted genetic modifications to improve Bt strains (Sun et al., 2016). 6.3 Multi-omics approaches for Bt strain optimization Multi-omics approaches integrate data from genomics, transcriptomics, proteomics, and metabolomics to provide a comprehensive understanding of the biological systems at play. For Bt strain optimization, this holistic view can reveal how different genes, proteins, and metabolic pathways interact to produce insecticidal toxins. By analyzing these interactions, researchers can identify key regulatory nodes and pathways that can be manipulated to enhance the efficacy of Bt strains. Transcriptomic analysis can reveal which genes are upregulated during toxin production, while proteomic studies can identify the proteins involved in this process. Metabolomic data can further elucidate the metabolic pathways that support toxin synthesis. Integrating these datasets allows for a more targeted and effective approach to Bt strain improvement (Wang et al., 2015). 7 Applications and Future Directions 7.1 Bt crops and sustainable agriculture Bt crops have significantly contributed to sustainable agriculture by reducing the reliance on chemical pesticides and enhancing pest management. The integration of Bacillus thuringiensis (Bt) genes into crops like maize, cotton, and soybean has provided effective control against major pests, leading to environmental and economic benefits (Xiao and Wu, 2019). However, the evolution of resistance in some pest populations poses a challenge to the long-term sustainability of Bt crops. Strategies such as the use of refuges of non-Bt host plants and the development of crops expressing multiple Bt genes are essential to delay resistance and maintain the efficacy of Bt crops. Additionally, the environmental safety of Bt crops has been well-documented, with studies showing no adverse effects on non-target organisms, further supporting their role in sustainable agriculture (Romeis et al., 2019). 7.2 Integration of Bt toxins with other control methods To enhance the effectiveness of Bt crops and manage resistance, integrating Bt toxins with other pest control methods is crucial. This integrated pest management (IPM) approach includes the use of biological control agents, crop rotation, and the application of other biopesticides (Gassmann and Reisig, 2022). By combining these methods, the pressure on pests to develop resistance to Bt toxins can be reduced, thereby prolonging the effectiveness of Bt crops. Moreover, the conservation of natural enemies through reduced use of chemical insecticides in Bt crop fields can further enhance pest control and contribute to a more balanced ecosystem. 7.3 Emerging trends in Bt genomic research Recent advancements in Bt genomic research are paving the way for the development of next-generation Bt crops. Researchers are focusing on understanding the molecular mechanisms of Bt toxin resistance in pests, which involves studying mutations in genes related to toxin activation, binding, and insect immune responses (Jurat-Fuentes et al., 2021). This knowledge is crucial for engineering novel Bt toxins with enhanced efficacy and reduced likelihood of resistance development. Additionally, the creation of chimeric Bt proteins by combining domains from different Cry proteins is an emerging trend that holds promise for producing more potent insecticidal proteins (Koch et al., 2015). Future research will likely explore the use of Bt crops in combination with other genetic technologies, such as RNA interference (RNAi), to target a broader range of pests and further improve pest management strategies. Acknowledgments I appreciate Dr Wang F. from the Hainan Institution of Biotechnology for her assistance in references collection and discussion for this work completion. Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.
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Bt Research 2024, Vol.15, No.5, 223-231 http://microbescipublisher.com/index.php/bt 223 Review Article Open Access Public Health Implications of Bt-based Mosquito Control Programs Minsheng Lin Hainan Tropical Agricultural Resources Research Institute, Tropical Microbial Resources Research Center, Sanya, 572025, Hainan, China Corresponding email: minsheng.lin@hitar.org Bt Research, 2024, Vol.15, No.5 doi: 10.5376/bt.2024.15.0022 Received: 27 Jul., 2024 Accepted: 06 Sep., 2024 Published: 21 Sep., 2024 Copyright © 2024 Lin, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Lin M.S., 2024, Public health implications of Bt-based mosquito control programs, Bt Research, 15(5): 223-231 (doi: 10.5376/bt.2024.15.0022) Abstract This study explores the public health implications of the use of Bacillus thuringiensis (Bt), especially Bacillus thuringiensis subsp. Israel (Bti), in mosquito control programs. Effective vector control is critical as mosquito-borne diseases such as malaria, dengue, Zika and West Nile virus pose increasing challenges. Bti-based interventions are a viable alternative to chemical insecticides due to their specificity, small environmental impact, and low propensity to develop resistance in target mosquito populations. The study illustrates the mechanism of action, efficacy, and environmental considerations of Bt toxin, as well as the potential for resistance development. Field studies and case studies highlight significant reductions in mosquito populations and disease incidence, confirming the effectiveness of Bt-based strategies in different ecological environments. Keywords Bacillus thuringiensis subsp. israel; Mosquito control; Mosquito-borne diseases; Environmental impacts; Resistance management 1 Introduction Bacillus thuringiensis (Bt) is a soil-dwelling bacterium that produces crystal proteins (Cry and Cyt toxins) during sporulation, which are highly toxic to various insect larvae, including mosquitoes. Bt subspecies israelensis (Bti) is particularly effective against mosquito larvae and has been widely used in mosquito control programs. The Cry and Cyt toxins from Bti disrupt the gut cells of mosquito larvae, leading to their death. Recent advancements have explored the synergistic effects of combining Bt toxins with other bacterial metabolites to enhance their efficacy against mosquito species such as Aedes albopictus and Culex pipiens pallens (Park et al., 2016). Additionally, novel formulations and genetic modifications, such as the expression of Bt toxins in green algae, have been developed to improve the sustainability and effectiveness of Bt-based mosquito control (Kang et al., 2017). Mosquito-borne diseases, including malaria, dengue, Zika, and West Nile virus, pose significant public health challenges globally. These diseases result in substantial morbidity and mortality, particularly in tropical and subtropical regions. Effective mosquito control is crucial in reducing the transmission of these diseases. Traditional chemical insecticides have faced issues such as resistance development and environmental concerns, making Bt-based biopesticides an attractive alternative due to their specificity and lower environmental impact2 6. However, the persistence of Bt toxins in the environment and potential resistance development in mosquito populations necessitate continuous monitoring and evaluation (Dorsch et al., 2002; Paris et al., 2011). This study comprehensively analyzes the impact of Bt based mosquito control programs on public health, covering the mechanism of action of Bt toxins, the effectiveness and environmental impact of Bt formulations, and the potential for mosquito population resistance development; We will also discuss the socio-economic aspects of mosquito control and the public's views on Bt based intervention measures. To provide information on sustainable and effective strategies for using Bt in mosquito control, while addressing the benefits and challenges associated with its application. 2 Bt-Based Mosquito Control Programs 2.1 Bt toxins and their mode of action on mosquito larvae Bacillus thuringiensis (Bt) toxins, particularly those fromBacillus thuringiensis var. israelensis (Bti), are highly effective in controlling mosquito larvae. These toxins work by producing crystal proteins (Cry and Cyt toxins) that
Bt Research 2024, Vol.15, No.5, 223-231 http://microbescipublisher.com/index.php/bt 224 target the midgut cells of mosquito larvae. The Cry toxins, such as Cry4Aa, Cry4Ba, and Cry11Aa, bind to specific receptors in the midgut, causing cell lysis and larval death. Cyt1Aa, another crucial toxin, acts synergistically with Cry toxins by inserting itself into the cell membrane, enhancing the overall insecticidal effect and reducing the risk of resistance development (Hayakawa et al., 2017; Silva-Filha et al., 2021). 2.2 Key Bt strains used in mosquito control The primary Bt strain used in mosquito control is Bacillus thuringiensis var. israelensis (Bti). Bti is favored due to its high specificity and effectiveness against mosquito larvae, including species such as Aedes aegypti and Culex pipiens. Other strains like Bacillus sphaericus are also used, known for their persistence in field conditions. Recent advancements have seen the development of formulations combining Bti with other bacterial strains or secondary metabolites to enhance efficacy and reduce resistance (Park et al., 2016; Derua et al., 2018). 2.3 Historical development and global implementation of bt-based mosquito control programs The use of Bt-based mosquito control programs began in the late 20th century, with Bti being introduced as a microbial larvicide. Over the years, Bti has been widely adopted globally due to its environmental safety and effectiveness. Programs have been implemented in various regions, including the highlands of western Kenya, where long-lasting formulations like FourStar® and LL3 have been used to control malaria vectors without significantly impacting non-target organisms. Additionally, Bt-based control methods have been integrated into broader public health strategies to combat mosquito-borne diseases, demonstrating significant reductions in mosquito populations and disease transmission (Allgeier et al., 2019). 3 Efficacy of Bt-Based Control Programs in Reducing Mosquito Populations 3.1 Field trials and case studies in urban and rural areas 3.1.1 Successes in dengue and malaria control programs Bt-based mosquito control programs have shown significant success in various field trials and case studies. A community-based application of Bacillus thuringiensis var. israelensis (Bti) in Rwanda demonstrated a substantial reduction in Anopheles larval habitats, with a 49% reduction in supervised areas and a 28% reduction in community-led areas. This intervention also nearly eliminated pupal production, thereby preventing the emergence of adult mosquitoes (Hakizimana et al., 2022). Similarly, a large-scale field trial in Burkina Faso using Bti for biological larviciding achieved a 77.4% reduction in adult Anopheles mosquito abundance, indicating the potential of Bt-based methods in malaria vector control (Dambach et al., 2020). 3.1.2 Variability in efficacy based on environmental conditions The efficacy of Bt-based mosquito control programs can vary significantly based on environmental conditions. Factors such as seasonality, local climate, and the specific ecological characteristics of the intervention area can influence outcomes. For example, the effectiveness of biological methods like Wolbachia-infected mosquitoes can be hampered by extreme weather conditions such as heatwaves, which may result in the loss of Wolbachia infection (Ogunlade et al., 2023). Additionally, the success of community-based interventions may depend on the level of community engagement and the availability of resources for sustained implementation (Figure 1) (Salazar et al., 2019). 3.2 Comparative effectiveness: Bt-based vs. chemical-based mosquito control When comparing Bt-based mosquito control methods to chemical-based approaches, several studies highlight the advantages of biological methods. Chemical methods, while initially effective, often lead to increased resistance in mosquito populations, reducing long-term efficacy (Ogunlade et al., 2023). In contrast, Bt-based methods, such as the use of Bti, have shown sustained effectiveness without the risk of resistance development. A systematic review and meta-analysis found that environmental methods, including the use of Bti, were more sustainable and posed fewer risks of environmental contamination compared to chemical insecticides (Buhler et al., 2019). Moreover, the use of Wolbachia-infected mosquitoes has demonstrated a significant reduction in dengue incidence, with a protective efficacy of 77.1% in treated areas (Utarini et al., 2021).
Bt Research 2024, Vol.15, No.5, 223-231 http://microbescipublisher.com/index.php/bt 225 Figure 1 (A) Location of the experimental sites in Ruhuha, southeast Rwanda. (B) Training of community representatives for spraying of Bti. (C) Spraying of Bti using knapsack sprayers in the irrigated rice fields (Adopted from Hakizimana et al., 2022) 3.3 Limitations and challenges in sustaining efficacy Despite the promising results, there are several limitations and challenges in sustaining the efficacy of Bt-based mosquito control programs. One major challenge is the need for continuous and consistent application to maintain effectiveness. The long-term success of biological larviciding with Bti requires regular treatments to ensure that mosquito populations do not rebound (Dambach et al., 2020). The variability in environmental conditions can affect the stability and persistence of biological agents like Wolbachia, necessitating ongoing monitoring and adaptation of strategies. Community-based programs may face challenges related to resource allocation, community participation, and the scalability of interventions (Hladish et al., 2018). Addressing these challenges is crucial for the long-term success and sustainability of Bt-based mosquito control programs. 4 Public Health Implications of Bt-Based Mosquito Control 4.1 Impact on mosquito-borne disease transmission Bt-based mosquito control programs have shown significant potential in reducing the transmission of mosquito-borne diseases such as dengue, malaria, and Zika virus. Studies have demonstrated that targeted biological interventions, including the use of Bacillus thuringiensis (Bt), can effectively reduce mosquito populations and subsequently lower disease incidence. For instance, a systematic review of cluster randomized controlled trials (cRCTs) highlighted that interventions like mass adult trapping and source reduction were associated with statistically significant reductions in disease transmission and entomological indicators (Oliver et al., 2021). Additionally, the deployment of Wolbachia-infected mosquitoes, which is another biological control strategy, has shown promise in reducing the transmission of dengue, Zika, and chikungunya viruses in field trials
Bt Research 2024, Vol.15, No.5, 223-231 http://microbescipublisher.com/index.php/bt 226 (Anders et al., 2018). These findings underscore the importance of integrating Bt-based methods into broader mosquito control programs to achieve substantial public health benefits. 4.2 Safety and environmental considerations Bt-based mosquito control is considered an eco-friendly alternative to traditional chemical insecticides, which often pose risks to human health and the environment. The use of Bt and other biological agents targets mosquito larvae specifically, minimizing the impact on non-target species and reducing the likelihood of environmental contamination. This approach addresses the growing concern over insecticide resistance, which has been a significant limitation of chemical control methods (Benelli et al., 2016). The application of Bt-based strategies aligns with the principles of integrated vector management, promoting sustainable and environmentally responsible practices (Achee et al., 2019). The safety profile of Bt-based interventions makes them a preferable option for communities that are wary of the adverse effects associated with chemical insecticides (Oliver et al., 2021). 4.3 Socio-economic benefits of Bt-based mosquito control The socio-economic benefits of Bt-based mosquito control programs are multifaceted. By reducing the incidence of mosquito-borne diseases, these programs can alleviate the healthcare burden on affected communities, leading to lower medical costs and improved public health outcomes. For example, the implementation of Wolbachia-infected mosquito deployments has been shown to be cost-effective in reducing dengue incidence, which can translate to significant economic savings for healthcare systems (Anders et al., 2018). Bt-based interventions can enhance community engagement and participation, as they are often perceived as safer and more acceptable compared to chemical spraying. This increased community involvement can lead to more effective and sustainable mosquito control efforts, further amplifying the socio-economic benefits (Jiang, 2024). Overall, Bt-based mosquito control programs offer a promising avenue for improving public health and economic stability in regions plagued by mosquito-borne diseases. 5 Challenges and Risks in Bt-Based Mosquito Control 5.1 Development of mosquito resistance to Bt toxins One of the primary challenges in Bt-based mosquito control programs is the potential development of resistance in mosquito populations. Bacillus thuringiensis israelensis (Bti) produces multiple toxins that target mosquito larvae, but continuous exposure can lead to the selection of resistant individuals. Studies have shown that resistance to individual Bti toxins can develop after only a few generations, although resistance to the complete Bti toxin mixture remains relatively low (Paris et al., 2011). The genetic basis of this resistance is complex, involving multiple genes and mechanisms, which complicates efforts to monitor and manage resistance (Bonin et al., 2015). Additionally, the persistence of Bti in the environment can impose continuous selective pressure, further accelerating the evolution of resistance (Tetreau et al., 2012). 5.2 Challenges in large-scale implementation Implementing Bt-based mosquito control on a large scale presents several logistical and operational challenges. The effectiveness of Bti can be influenced by environmental factors such as temperature, pH, and organic matter, which can affect the stability and activity of the toxins (Silva-Filha et al., 2021). Moreover, achieving uniform distribution of Bti in diverse and often inaccessible breeding habitats of mosquitoes is difficult. The need for repeated applications to maintain effective control adds to the complexity and cost of large-scale operations. Additionally, integrating Bti with other control measures, such as synthetic predator cues, requires careful planning and coordination to ensure synergistic effects and avoid unintended consequences (Beeck et al., 2016). 5.3 Regulatory and policy challenges Regulatory and policy challenges also pose significant hurdles to the widespread adoption of Bt-based mosquito control programs. The approval process for biopesticides can be lengthy and complex, involving rigorous testing to ensure safety and efficacy. There is also a need for clear guidelines and standards for the production, application, and monitoring of Bti to prevent misuse and resistance development (Jurat-Fuentes et al., 2021). Public
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