Prospects and Pitfalls: Next-Generation Tools to Control Mosquito-Transmitted Disease.

Mosquito-transmitted diseases, including malaria and dengue, are a major threat to human health around the globe, affecting millions each year. A diverse array of next-generation tools has been designed to eliminate mosquito populations or to replace them with mosquitoes that are less capable of transmitting key pathogens. Many of these new approaches have been built on recent advances in CRISPR/Cas9-based genome editing. These initiatives have driven the development of pathogen-resistant lines, new genetics-based sexing methods, and new methods of driving desirable genetic traits into mosquito populations. Many other emerging tools involve microorganisms, including two strategies involving Wolbachia that are achieving great success in the field. At the same time, other mosquito-associated bacteria, fungi, and even viruses represent untapped sources of new mosquitocidal or antipathogen compounds. Although there are still hurdles to be overcome, the prospect that such approaches will reduce the impact of these diseases is highly encouraging.

Protein complexes from edible mushrooms as a sustainable potato protection against coleopteran pests.

Protein complexes from edible oyster mushrooms (Pleurotus sp.) composed of pleurotolysin A2 (PlyA2) and pleurotolysin B (PlyB) exert toxicity in feeding tests against Colorado potato beetle (CPB) larvae, acting through the interaction with insect-specific membrane sphingolipid. Here we present a new strategy for crop protection, based on in planta production of PlyA2/PlyB protein complexes, and we exemplify this strategy in construction of transgenic potato plants of cv Désirée. The transgenics in which PlyA2 was directed to the vacuole and PlyB to the endoplasmic reticulum are effectively protected from infestation by CPB larvae without impacting plant performance. These transgenic plants showed a pronounced effect on larval feeding rate, the larvae feeding on transgenic plants being on average five to six folds lighter than larvae feeding on controls. Further, only a fraction (11%-37%) of the larvae that fed on transgenic potato plants completed their life cycle and developed into adult beetles. Moreover, gene expression analysis of CPB larvae exposed to PlyA2/PlyB complexes revealed the response indicative of a general stress status of larvae and no evidence of possibility of developing resistance due to the functional inactivation of PlyA2/PlyB sphingolipid receptors.

Current status and future trends of microbial and nematode-based biopesticides for biocontrol of crop pathogens.

The increasing public demand to avoid the use of synthetic pesticides and fertilizers in agricultural production systems, causing serious environmental damages, has challenged industry to develop new and effective solutions to manage and control phytopathogens. Biopesticides, particularly microbial-based biopesticides, are a promising new alternative with high biodegradability, specificity, suitability for incorporation into integrated pest management practices, low likelihood of resistance development, and practically no known human health risks. However: expensive production methods, narrow action spectra, susceptibility to environmental conditions, short shelf life, poor storage stability, legislation registry constraints, and general lack of knowledge are slowing down their adoption. In addition to regulatory framework revisions and improved training initiatives, improved preservation methods, thoughtfully designed formulations, and field test validations are needed to offer new microbial- and nematode-based biopesticides with improved efficacy and increased shelf-life. During the last several years, substantial advancements in biopesticide production have been developed. The novelty part of this review written in 2023 is to summarize (i) mechanisms of action of beneficial microorganisms used to increase crop performance and (ii) successful formulation including commercial products for the biological control of phytopathogens based on microorganisms, nematode and/or metabolites.

Total synthesis of antifungal lipopeptide iturin A analogues and evaluation of their bioactivity against F. graminearum.

The pursuit of novel antifungal agents is imperative to tackle the threat of antifungal resistance, which poses major risks to both human health and to food security. Iturin A is a cyclic lipopeptide, produced by Bacillus sp., with pronounced antifungal properties against several pathogens. Its challenging synthesis, mainly due to the laborious synthesis of the ß-amino fatty acid present in its structure, has hindered the study of its mode of action and the development of more potent analogues. In this work, a facile synthesis of bioactive iturin A analogues containing an alkylated cysteine residue is presented. Two analogues with opposite configurations of the alkylated cysteine residue were synthesized, to evaluate the role of the stereochemistry of the newly introduced amino acid on the bioactivity. Antifungal assays, conducted against F. graminearum, showed that the novel analogues are bioactive and can be used as a synthetic model for the design of new analogues and in structure-activity relationship studies. The assays also highlight the importance of the ß-amino acid in the natural structure and the role of the stereochemistry of the amino fatty acid, as the analogue with the D configuration showed stronger antifungal properties than the one with the L configuration.

A novel spider venom peptide from the predatory mite Neoseiulus barkeri shows lethal effect on phytophagous pests.

The long-term use of pesticides in the field, and the high fertility and adaptability of phytophagous mites have led to resistance problems; consequently, novel safe and efficient active substances are necessary to broaden the tools of pest mite control. Natural enemies of arthropods typically secrete substances with paralytic or lethal effects on their prey, and those substances are a resource for future biopesticides. In this study, two putative venom peptide genes were identified in a parasitic mite Neoseiulus barkeri transcriptome. Recombinant venom NbSP2 peptide injected into Tetranychus cinnabarinus mites was significantly more lethal than recombinant NBSP1. NbSP2 was also lethal to Spodoptera litura when injected but not when fed to third instar larvae. The interaction proteins of NbSP2 in T. cinnabarinus and S. litura were identified by affinity chromatography. Among these proteins, ATP synthase subunit ß (ATP SSß) was deduced as a potential target. Four binding sites were predicted between NBSP2 and ATP SSß of T. cinnabarinus and S. litura. In conclusion, we identified a venom peptide with activity against T. cinnabarinus and S. litura. This study provides a novel component for development of a new biological pesticide.

Essential oils for managing anthracnose in mango (Mangifera indica): laboratory results do not translate into field efficacy.

Essential oil-based products with broad plant disease control claims are commercially available and may be a practical alternative to copper fungicides for crop protection in organic mango orchards. We evaluated the disease control efficacy and crop safety of thyme oil, savory oil, and tree tea oil through replicated in vitro, in vivo (detached leaf and potted trees), and field assays. Three Colletotrichum species associated with mango anthracnose were tested in vitro, whereas only C. siamense was used for in vivo assays. Within the range of concentrations tested in vitro (62.5 to 2,000 µl a.i./liter), thyme and savory oil displayed fungicidal activity, whereas no fungistatic or fungicidal activity was observed with tea tree oil. In the in vivo assays, none of the treatments based on a preventive application rate of thyme (1,150 µl a.i./liter), savory (2,000 µl a.i./liter), or tea tree oil (342 µl a.i./liter) were effective in preventing the development of anthracnose on wounded and artificially inoculated leaves. Although field applications of thyme or tea tree oil did not result in phytotoxicity or negative impacts on fruit yield, they were ineffective in reducing the incidence and severity of naturally occurring anthracnose. Applications of copper hydroxide approved for organic agriculture were effective in controlling anthracnose in the field, and no added benefits were found by premixing this compound with thyme oil. Results indicate that essential oil products based on thyme or tea tree oil are inefficient at controlling anthracnose in mangoes.

Hesperidin as a Species-Specific Modifier of Aphid Behavior.

Hesperidin is a highly bioactive natural flavonoid whose role in ecological interactions is poorly known. In particular, the effects of hesperidin on herbivores are rarely reported. Flavonoids have been considered as prospective biopesticides; therefore, the aim of the present study was to examine the influence of hesperidin on the host plant selection behavior of three aphid (Hemiptera: Aphididae) species: Acyrthosiphon pisum Harrris, Rhopalosiphum padi (L.), and Myzus persicae (Sulz.). The aphid host plants were treated with 0.1% and 0.5% ethanolic solutions of hesperidin. Aphid probing behavior in the no-choice experiment was monitored using electropenetrography and aphid settling on plants in the choice experiment was recorded. The results demonstrated that hesperidin can be applied as a pre-ingestive, ingestive, and post-ingestive deterrent against A. pisum, as an ingestive deterrent against R. padi, and as a post-ingestive deterrent against M. persicae using the relatively low 0.1% concentration. While in A. pisum the deterrent effects of hesperidin were manifested as early as during aphid probing in peripheral plant tissues, in M. persicae, the avoidance of plants was probably the consequence of consuming the hesperidin-containing phloem sap.

Chalcones-Features, Identification Techniques, Attributes, and Application in Agriculture.

This review article is a comprehensive and current overview on chalcones, covering their sources, identification methods, and properties with a particular focus on their applications in the agricultural sector. The widespread use of synthetic pesticides has not only led to increased resistance among weeds and pests, resulting in economic losses, but it has also raised significant health concerns due to the overuse of these chemicals. In line with the European Green Deal 2030 and its Farm to Fork strategy, there is a targeted 50% reduction in the use of chemical pesticides by 2030, emphasizing a shift towards natural alternatives that are more environmentally sustainable and help in the restoration of natural resources. Chalcones and their derivatives, with their herbicidal, fungicidal, bactericidal, and antiviral properties, appear to be ideal candidates. These naturally occurring compounds have been recognized for their beneficial health effects for many years and have applications across multiple areas. This review not only complements the previous literature on the agricultural use of chalcones but also provides updates and introduces methods of detection such as chromatography and MALDI technique.

Is the Biopesticide from Tea Tree Oil an Effective and Low-Risk Alternative to Chemical Pesticides? A Critical Review.

The use of chemical pesticides in agriculture contributes to soil, water and air pollution, biodiversity loss, and injury to non-target species. The European Commission has already established a Harmonized Risk Indicator to quantify the progress in reducing the risks linked to pesticides. Therefore, there is an increasing need to promote biopesticides, or so-called low-risk pesticides (LRP). Tea tree oil (TTO) is known for its antiseptic, antimicrobial, antiviral, antifungal, and anti-inflammatory properties. TTO has been extensively studied in pest management as well as in the pharmaceutical and cosmetic industry; there are already products based on its active substances on the market. This review focuses on the overall evaluation of TTO in terms of effectiveness and safety as a biopesticide for the first time. The collected data can be an added value for further evaluation of TTO in terms of the authorization extension as a fungicide in 2026.

Valorisation of essential oil of Eucalyptus populifolia Desf, Eucalyptus woollsiana and Eucalyptus exserta for agro-industrial purposes.

Forest species are characterized by their wealth of essential oils (EOs), which play key requested for alternative control methods against weeds, fungi and pests. This study reports the chemical composition and highlight the antioxidant, antifungal and phytotoxic properties of the EOs obtained from Eucalyptus populifolia Desf, Eucalyptus woollsiana and Eucalyptus exserta. The EOs were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Their antioxidant, antifungal and phytotoxic properties were investigated. All EOs displayed a specific richness in eucalyptol (75.94-7.01%), camphene (6.97-0.4%) globulol (5.20-5.83%) and α-pinene (0.71-12.82 %). EOs isolated from E. populifolia exhibited significant antioxidant activity. Furthermore, all investigated EOs displayed significant antifungal properties against four phytopathogenic fungi belonging to Fusarium genus. The phytotoxic activity against five weeds showed varying effectiveness on seed germination and seedling growth, which exhibited greater effectiveness compared to glyphosate. Eucalyptus EOs have the potential to be used in the formulation of biopesticides.

Pesticides and winemaking: A comprehensive review of conventional and emerging approaches.

The use of pesticides in viticulture may play a crucial role in ensuring the health and quality of grapes. This review analyzes the most common pesticides used, illustrating their classification and toxicity, and their variations throughout the winemaking process. Fungicides are generally harmless or mildly toxic, whereas insecticides are classified as either highly or moderately hazardous. Potential alternatives to synthetic pesticides in wine production are also reviewed, thereby including biopesticides and biological agents. Analytical methods for detecting and quantifying pesticide residues in wine are then described, including liquid chromatography and gas chromatography coupled with mass spectrometry. This review also discusses the impact of the winemaking process on pesticide content. Pesticides with strong hydrophobicity were more likely to accumulate in solid byproducts, whereas hydrophilic pesticides were distributed more in the liquid phase. Grape's skin contains lipids, so hydrophobic pesticides adsorb strongly on grape surfaces and the clarification has been shown to be effective in the reduction of hydrophobic compounds. Therefore, the final wine could have more quantities of hydrophilic pesticides. Alcoholic fermentation has been shown to be crucial in pesticide dissipation. However, pesticide residues in wine have been shown an antagonistic effect on yeasts, affecting the safety and quality of wine products. Therefore, proteomic and genomic analyses of yeast growth are reviewed to understand the effects of pesticides on yeast during fermentation. The last section describes new effective methods used in removing pesticides from grapes and wine, thereby improving product quality and reducing harmful effects.

Recent advances in Bacillus-mediated plant growth enhancement: a paradigm shift in redefining crop resilience.

The Bacillus genus has emerged as an important player in modern agriculture, revolutionizing plant growth promotion through recent advances. This review provides a comprehensive overview of the critical role Bacillus species play in boosting plant growth and agricultural sustainability. Bacillus genus bacteria benefit plants in a variety of ways, according to new research. Nitrogen fixation, phosphate solubilization, siderophore production, and the production of growth hormones are examples of these. Bacillus species are also well-known for their ability to act as biocontrol agents, reducing phytopathogens and protecting plants from disease. Molecular biology advances have increased our understanding of the complex interplay between Bacillus species and plants, shedding light on the genetic and metabolic underpinnings of these interactions. Furthermore, novel biotechnology techniques have enabled the development of Bacillus-based biofertilizers and biopesticides, providing sustainable alternatives to conventional chemical inputs. Apart from this, the combination of biochar and Bacillus species in current biotechnology is critical for improving soil fertility and encouraging sustainable agriculture through enhanced nutrient retention and plant growth. This review also emphasizes the Bacillus genus bacteria's ability to alleviate environmental abiotic stresses such as drought and salinity, hence contributing to climate-resilient agriculture. Moreover, the authors discuss the challenges and prospects associated with the practical application of Bacillus-based solutions in the field. Finally, recent advances in Bacillus-mediated plant growth promotion highlight their critical significance in sustainable agriculture. Understanding these improvements is critical for realizing the full potential of Bacillus genus microorganisms to address current global food production concerns.

The possibility of using Serratia isolates for the production of biopreparations in the protection of plants against diseases and pests.

The agriculture is extremely important for the life of human beings. Therefore, it is needed to control the enemies that destroy vast areas of crops causing great economic losses. Chemical pesticides were the option for many decades, but the damage that they cause to environment and human health led to the idea of changing the use of these for more sustainable options such as biopesticides as a biological control. Among microbial pesticides, Serratia species have been found as suitable options to apply against several pests or pathogens. Serratia species produce a wide range of secondary metabolites with several biological activities such as antifungal, antibacterial, and pesticides which can be used in sustainable agriculture. It has been reported that several Serratia species are able to suppress some crop diseases caused by Fusarium oxysporum, Rhizoctonia solani, Phytophthora parasitica, Sclerotinia sclerotiorum, Verticillium dahlia, and Phytophthora capsici among others. Therefore, they have been used as biocontrol agents in agriculture. In this review, we summarized the genus Serratia describing its history and development and the metabolites it secretes, which are responsible for their antibacterial and antifungal activity. We have analyzed the insecticide capacity of several Serratia species as well antifungal properties of Serratia species against most important crops' pathogens. In conclusion, the use of Serratia as a biological control agent against plant pathogens can be a good option for a sustainable agriculture. More work is needed to assess the safety of the isolated new strains and their effectiveness against pathogens in in vivo conditions.

Lactic acid bacteria and Bacillus spp. as fungal biological control agents.

Fungal pathogens are one of the most important agents affecting crop production and food safety, and agrochemical application is one of the main approaches to reduce phytopathogenic fungi contamination in agricultural products. However, excessive and inadequate use can cause environmental damage, human and animal hazard, and increased phytopathogen resistance to fungicides. Biological control using lactic acid bacteria (LAB) and Bacillus spp. is an environmentally friendly strategy for phytopathogenic fungi management. Several molecules produced by these bacteria indeed affect fungal growth and viability in different plant crops. In this article, the activity spectra are reviewed along with the antifungal effect and antifungal compounds produced by LAB (e.g. organic acids, peptides, cyclic dipeptides, fatty acids, and volatile compounds) and Bacillus spp. (e.g. peptides, enzymes, and volatile compounds).

Phytochemical profiling, antioxidant activities, enzymatic activities and insecticidal potential of aqueous extracts of four plants on the larvae of Helicoverpa armigera (Lepidoptera: Noctuidae), the main pest of cotton plant in Ivory Coast.

In the context of climate change, the Ivorian cotton industry is facing with the loss of sensitivity of pests (Helicoverpa armigera) and the appearance of new so-called emerging insects. Faced with this situation, cotton producers tend to use insecticide products in high doses, in excess of the norm. However, the misuse of chemical products poses many health risks. Therefore, to limit the use of chemicals, aqueous extracts of local plants with insecticidal properties were examined in the laboratory and in the field. Four local plant species were selected [Anacardium occidentale (Anarcardier); Azadirachta indica (Neem); Hyptis suaveolens (Hyptis) and Tephrosia vogelii (Tephrosia)]. After determining the chemical profiles of the four extracts by high performance liquid chromatography (HPLC)-mass spectrometry, their inhibitory activities were assessed in cholinesterase and tyrosinase. The sensitivity of Helicoverpa armigera larvae was evaluated by ingesting the aqueous extracts at several concentrations ranging from 2% to 64% in an artificial nutrient substrate. Then, the mortality rates of the larvae during 72 h were evaluated and the lethal concentrations were determined. The results of chemical analyses (HPLC) showed that the richest aqueous extract in phytochemicals with 54 elements detected was that of cashew (A. occidentale). T. vogelii, A. indica and H. suaveolens presented 44, 45, and 39 chemical compounds, respectively. In addition, the total phenolic content was higher in A. occidentale (110.67 mg gallic acid equivalents/g) followed by A. indica (42.43 mg gallic acid equivalents/g). The highest antioxidant ability was observed with the aqueous extract of cashew (A. occidentale). Anti-enzymatic activities such as acetylcholinesterase, butyrylcholinesterase and tyrosinase inhibition were most pronounced in A. occidentale (2.35 ± 0.02 mg galanthamine equivalent/g, 3.77 ± 0.01 mg galanthamine equivalent/g and 71.28 ± 0.07 mg kojic acid equivalent/g, respectively). The most toxic aqueous extract for H. armigera larvae was that of cashew with a lethal concentration LC50 = 11.68%. Moreover, the principal component analysis performed showed that the insecticidal activity is strongly correlated with the antioxidant and enzymatic activities of the aqueous extracts. Then, the hierarchical ascending classification showed cashew as the best plant. For the sustainability of cotton production, it would be necessary to limit the use of chemical-synthetic insecticides through the use of plant extracts, especially from cashew leaves.

Control efficacy of azadirachtin on the fall armyworm, Spodoptera frugiperda (J. E. Smith) by soil drenching.

The fall armyworm (FAW), Spodoptera frugiperda, is an important agricultural pest species native to the Western Hemisphere and has recently invaded to Africa and Asia. Owing to the development of pesticide resistance and environmental contamination, ecofriendly pesticides are desirable for FAW control. Azadirachtin is a plant-derived natural pesticide with low toxicity to humans and the natural environment. Azadirachtin is primarily applied by foliar spraying; however, this approach lowers the efficacy of controlling target insects owing to photodegradation and might give a harmful effect on nontarget beneficial insects. Thus, we investigated whether applying azadirachtin to soil improves FAW control and its toxicity to corn plants. Soil drainage of azadirachtin exhibited no phytotoxic effects on corn plants but significantly reduced the larval body weight and delayed the developmental period of each larval instar of FAW. Applying 10, 15, and 20 ppm azadirachtin to soil inhibited larval growth by 68%, 76%, and 91%, respectively. Furthermore, the survival rate of FAW gradually decreased when larvae were fed azadirachtin-treated corn leaves. Collectively, this is the first study suggesting the systemic efficacy of azadirachtin by soil drenching against FAW.

The use of RNA interference for the management of arthropod pests in livestock farms.

Pest management in farm animals is an important action to contain economic damage to livestock production and prevent transmission of severe diseases to the stock. The use of chemical insecticides is still the most common approach followed by farmers; however, avoiding possible toxic effects on animals is a fundamental task for pest control measures compatible with animal well-being. Moreover, legal constraints and insurgence of resistance by target species to the available insecticidal compounds are increasingly complicating farmers' operations. Alternatives to chemical pesticides have been explored with some promising results in the area of biological control or the use of natural products as sprays. The application of RNA interference techniques has enabled the production of new means of pest control in agriculture, and it is opening a promising avenue for controlling arthropod pests of livestock. Transcript depletion of specific target genes of the recipient organisms is based on the action of double-strand RNAs (dsRNA) capable of impairing the production of fundamental proteins. Their mode of action, based on the specific recognition of short genomic sequences, is expected to be highly selective towards non-target organisms potentially exposed; in addition, there are physical and chemical barriers to dsRNA uptake by mammalian cells that render these products practically innocuous for higher animals. Summarising existing literature on gene silencing for main taxa of arthropod pests of livestock (Acarina, Diptera, Blattoidea), this review explores the perspectives of practical applications of dsRNA-based pesticides against the main pests of farm animals. Knowledge gaps are summarised to stimulate additional research in this area.

Ecosystem services of entomopathogenic ascomycetes.

Entomopathogenic ascomycetes (EA) are an important part of the microbiota in most terrestrial ecosystems, where they can be found regulating natural populations of arthropod pests in both epigeous and hypogeous habitats while also establishing unique relationships with plants. These fungi offer direct benefits to agriculture and human welfare. In the present work, we conducted a systematic review to comprehensively assess the range of ecosystem services provided by EA, including direct and indirect pest biocontrol, plant growth promotion, plant defense against other biotic and abiotic stresses, nutrient cycling, and the production of new bioactive compounds with agricultural, pharmaceutical and medical importance. Moreover, EA are compatible with the ecosystem services provided by other microbial and macrobial biocontrol agents. This systematic review identified the need for future research to focus on evaluating the economic value of the ecological services provided by EA with a special emphasis on hypocrealean fungi. This evaluation is essential not only for the conservation but also for better regulation and exploitation of the benefits of EA in promoting agricultural sustainability, reducing the use of chemicals that enter the environment, and minimizing the negative impacts of crop protection on the carbon footprint and human health.

UV-C and wet heat resistance of Bacillus thuringiensis biopesticide endospores compared to foodborne Bacillus cereus endospores.

Bacillus endospores (spores) are generally resistant to environmental and food processing-related stress including thermal and non-thermal processing in the food industry, such as pasteurization, and UV-C inactivation. Bacillus thuringiensis insecticidal crystals and spores as the active substances in commercial biopesticides can also be introduced to vegetable foods and their food processing environment due to pre-harvest treatment of edible crops. The resistance of B. thuringiensis biopesticide spores in comparison to the genetically closely related foodborne B. cereus against heat and UV-C treatment is investigated in this study. The results show that B. thuringiensis biopesticide spores with the commercial granulated product formulation are better protected and as such more resistant to both wet heat (D values at 90 °C: 50.1-79.5 min) and UV-C treatment (D values at 0.6 mW/cm2: 7.5-8.9 min) than the pure spore suspension. The enhanced UV-C resistance properties of B. thuringiensis-formulated spores also indicate that the B. thuringiensis spores in powder or granule formulation applied in the field might not be effectively inactivated by solar radiation (UV-A and UV-B) in a short period. Furthermore, the spores of one emetic B. cereus toxin-producing strain (LFMFP 254; a Belgian outbreak strain) were found more resistant to the wet heat at 90 °C (D90-value = 71.2 min) than other tested pure spore suspensions, although the spores of B. cereus 254 did not show different behavior against UV-C treatment. This result suggests that UV-C treatment can be applied as an effective inactivation method against B. cereus 254 spores.

LmFKBP24 interacts with LmEaster to inhibit the antifungal immunity of Locusta migratoria.

Locusta migratoria is one of the most destructive pests that threaten crop growth and food production security in China. Metarhizium anisopliae has been widely used to control locusts around the world. Previous laboratory studies have revealed that LmFKBP24 is significantly upregulated after M. anisopliae infection, suggesting that it may play a role in immune regulation, yet the mechanism remains largely unknown. To gain further insight, we conducted an RNA interference (RNAi) study to investigate the function of LmFKBP24 in the regulation of antifungal immunity and analyzed the expression patterns of immune-induced genes. Our research revealed that LmFKBP24 is activated and upregulated when locusts are infected by M. anisopliae, and it inhibits the expression of antimicrobial peptide (AMP) defensin in the downstream of Toll pathway by combining with LmEaster rather than LmCyPA, thus exerting an immunosuppressive effect. To further investigate this, we conducted yeast two-hybrid (Y2H) and pull down assays to identify the proteins interacting with LmFKBP24. Our results provided compelling evidence for revealing the immune mechanism of L. migratoria and uncovered an innovative target for the development of new biological pesticides. Furthermore, our research indicates that LmFKBP24 interacts with LmEaster through its intact structure, providing a strong foundation for further exploration.