Improved control of Trialeurodes vaporariorum using mixture combinations of entomopathogenic fungi and the chemical insecticide spiromesifen.

Greenhouse whitefly (Trialeurodes vaporariorum) is a major global pest, causing direct damage to plants and transmitting viral plant diseases. Management of T. vaporariorum is problematic because of widespread pesticide resistance, and many greenhouse growers rely on biological control agents to regulate T. vaporariorum populations. However, these are often slow and vary in efficacy, leading to subsequent application of chemical insecticides when pest populations exceed threshold levels. Combining chemical and biological pesticides has great potential but can result in different outcomes, from positive to negative interactions. In this study, we evaluated co-applications of the entomopathogenic fungi (EPF) Beauveria bassiana and Cordyceps farinosa and the chemical insecticide spiromesifen in laboratory bioassays. Complex interactions between the EPFs and insecticide were described using an ecotoxicological mixtures model, the MixTox analysis. Depending on the EPF and chemical concentrations applied, mixtures resulted in additivity, synergism, or antagonism in terms of total whitefly mortality. Combinations of B. bassiana and spiromesifen, compared to single treatments, increased the rate of kill by 5 days. Results indicate the potential for combined applications of EPF and spiromesifen as an effective integrated pest management strategy and demonstrate the applicability of the MixTox model to describe complex mixture interactions.

Lysinibacillus pinottii sp. nov., a novel species with anti-mosquito and anti-mollusk activity.

An isolate of a Gram-positive, strictly aerobic, motile, rod-shaped, endospore forming bacterium was originally isolated from soil when screening and bioprospecting for plant beneficial microorganisms. Phylogenetic analysis of the 16S rRNA gene sequences indicated that this strain was closely related to Lysinibacillus fusiformis NRRL NRS-350T (99.7%) and Lysinibacillus sphaericus NRRL B-23268T (99.2%). In phenotypic characterization, the novel strain was found to grow between 10 and 45 °C and tolerate up to 8% (w/v) NaCl. Furthermore, the strain grew in media with pH 5 to 10 (optimal growth at pH 7.0). The predominant cellular fatty acids were observed to be iso-C15: 0 (52.3%), anteiso-C15: 0 (14.8%), C16:1ω7C alcohol (11.2%), and C16: 0 (9.5%). The cell-wall peptidoglycan contained lysine-aspartic acid, the same as congeners. A draft genome was assembled and the DNA G+C content was determined to be 37.1% (mol content). A phylogenomic analysis on the core genome of the new strain and 5 closest type strains of Lysinibacillus revealed this strain formed a distinct monophyletic clade with the nearest neighbor being Lysinibacillus fusiformis. DNA-DNA relatedness studies using in silico DNA-DNA hybridizations (DDH) showed this species was below the species threshold of 70%. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that this strain represents a novel species within the genus Lysinibacillus, for which the name Lysinibacillus pinottii sp. nov. is proposed, with type strain PB211T (= NRRL B-65672T, = CCUG 77181T).

Corrigendum: Essential Oils prime epigenetic and metabolomic changes in tomato defense against Fusarium oxysporum.

[This corrects the article DOI: 10.3389/fpls.2022.804104.].

An Ecofriendly Nature-Inspired Microcarrier for Enhancing Delivery, Stability, and Biocidal Efficacy of Phage-Based Biopesticides.

In pursuit of sustainable agricultural production, the development of environmentally friendly and effective biopesticides is essential to improve food security and environmental sustainability. Bacteriophages, as emerging biocontrol agents, offer an alternative to conventional antibiotics and synthetic chemical pesticides. The primary challenges in applying phage-based biopesticides in agricultural settings are their inherent fragility and low biocidal efficacy, particularly the susceptibility to sunlight exposure. This study addresses the aforementioned challenges by innovatively encapsulating phages in sporopollenin exine capsules (SECs), which are derived from plant pollen grains. The size of the apertures on SECs could be controlled through a non-thermal and rapid process, combining reinflation and vacuum infusion techniques. This unique feature facilitates the high-efficiency encapsulation and controlled release of phages under various conditions. The proposed SECs could encapsulate over 9 log PFU g-1 of phages and significantly enhance the ultraviolet (UV) resistance of phages, thereby ensuring their enhanced survivability and antimicrobial efficacy. The effectiveness of SECs encapsulated phages (T7@SECs) in preventing and treating bacterial contamination on lettuce leaves is further demonstrated, highlighting the practical applicability of this novel biopesticide in field applications. Overall, this study exploits the potential of SECs in the development of phage-based biopesticides, presenting a promising strategy to enhancing agricultural sustainability.

Comparative transcriptome analysis reveals disruption of Plutella xylostella immune system by fungal peptide cyclosporin C.

The diamondback moth (Plutella xylostella), a major threat to crucifers across the globe, has developed resistance against the majority of insecticides enhancing the need for alternate control measures against this pest. Recently cyclosporin C, a secondary metabolite produced by the insect pathogenic fungus Purpeocillium lilacinum, has been reported to induce lethal and sub-lethal effects against P. xylostella. To date, little is known about the molecular mechanisms of interaction between cyclosporin C and P. xylostella immune systems. This study reports the transcriptome-based immune response of P. xylostella to cyclosprin C treatment. Our results showed differential expression of 322, 97, and 504 differentially expressed genes (DEGS) in P. xylostella treated with cyclosporin C compared to control 24, 48, and 72 h post-treatment, respectively. Thirteen DEGs were commonly expressed at different time intervals in P. xylostella larvae treated with cyclosporin C compared to control. Cyclosporin C treatment induced the down-regulated expression of majority of immune-related genes related to pattern recognition responses, signal modulation, Toll and IMD pathways, antimicrobial peptides and antioxidant responses confirming the ability to suppress immune response of P. xylostella. These results will further improve our knowledge of the infection mechanism and complex biochemical processes involved in interaction between cyclosporin C and insect immune systems.

Enhancing the yield of Xenocoumacin 1 in Xenorhabdus nematophila YL001 by optimizing the fermentation process.

Xenocoumacin 1 (Xcn 1), antibiotic discovered from secondary metabolites of Xenorhabdus nematophila, had the potential to develop into a new pesticide due to its excellent activity against bacteria, oomycetes and fungi. However, the current low yield of Xcn1 limits its development and utilization. To improve the yield of Xcn1, response surface methodology was used to determine the optimal composition of fermentation medium and one factor at a time approach was utilized to optimize the fermentation process. The optimal medium composed of in g/L: proteose peptone 20.8; maltose 12.74; K2HPO4 3.77. The optimal fermentation conditions were that 25 °C, initial pH 7.0, inoculum size 10%, culture medium 75 mL in a 250 mL shake flask with an agitation rate of 150 rpm for 48 h. Xenorhabdus nematophila YL001 was produced the highest Xcn1 yield (173.99 mg/L) when arginine was added to the broth with 3 mmol/L at the 12th h. Compared with Tryptic Soy Broth medium, the optimized fermentation process resulted in a 243.38% increase in Xcn1 production. The obtained results confirmed that optimizing fermentation technology led to an increase in Xcn1 yield. This work would be helpful for efficient Xcn1 production and lay a foundation for its industrial production.

Phenotypic and genotypic characterization of fifty strains of Beauveria spp. (Ascomycota, Cordycipitaceae) fungal entomopathogens from diverse geographic origins against the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

BACKGROUND: The diamondback moth (DBM) (Plutella xylostella) causes large losses to global crop production. Conventional insecticides are losing effectiveness due to resistance. Consequently, there is a growing interest in sustainable control methods like entomopathogenic fungi (EPF) in Integrated Pest Management. However, the field efficacy of fungi varies due to environmental influences. In this study, a group of 50 Beauveria strains sourced from different locations were characterized by genotype and phenotype with respect to their conidial production, temperature and UV-B radiation tolerance, and virulence against DBM. RESULTS: Phylogenetic analysis revealed two distinct species: Beauveria bassiana (84%) and B. pseudobassiana (16%). Most strains showed optimal growth between 25 °C and 28 °C, with germination severely affected at 10 °C and 33 °C. Notably, 44% displayed high resistance to UV-B radiation (5.94 kJ m-2), with germination rates between 60.9% and 88.1%. Geographical origin showed no correlation with temperature or UV radiation tolerance. In virulence experiments, 52% of strains caused mortality rates exceeding 80% in DBM second instars at 7 days after exposure to a 4 mL conidial suspension (107 conidia/mL). CONCLUSION: Survival under environmental conditions is crucial for EPF-based commercial products against DBM. Results suggest strain tolerance to environmental stressors is more tied to specific micro-climatic factors than geographical origin. Each strain exhibited unique characteristics; for example, the most virulent strain (#29) was highly UV-sensitive. Therefore, characterizing diverse strains provides essential genotypic and phenotypic insights, which are fundamental for understanding their role as biocontrol agents while facilitating efficient biopesticide product development and uptake. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Mechanism Analysis of Antimicrobial Peptide NoPv1 Related to Potato Late Blight through a Computer-Aided Study.

Phytophthora infestans (Mont.) de Bary, the oomycotic pathogen responsible for potato late blight, is the most devastating disease of potato production. The primary pesticides used to control oomycosis are phenyl amide fungicides, which cause environmental pollution and toxic residues harmful to both human and animal health. To address this, an antimicrobial peptide, NoPv1, has been screened to target Plasmopara viticola cellulose synthase 2 (PvCesA2) to inhibit the growth of Phytophthora infestans (P. infestans). In this study, we employed AlphaFold2 to predict the three-dimensional structure of PvCesA2 along with NoPv peptides. Subsequently, utilizing computational methods, we dissected the interaction mechanism between PvCesA2 and these peptides. Based on this analysis, we performed a saturation mutation of NoPv1 and successfully obtained the double mutants DP1 and DP2 with a higher affinity for PvCesA2. Meanwhile, dynamics simulations revealed that both DP1 and DP2 utilize a mechanism akin to the barrel-stave model for penetrating the cell membrane. Furthermore, the predicted results showed that the antimicrobial activity of DP1 was superior to that of NoPv1 without being toxic to human cells. These findings may offer insights for advancing the development of eco-friendly pesticides targeting various oomycete diseases, including late blight.

RNA interference as a next-generation control method for suppressing Varroa destructor reproduction in honey bee (Apis mellifera) hives.

BACKGROUND: The Varroa mite (Varroa destructor) is considered to be the greatest threat to apiculture worldwide. RNA interference (RNAi) using double-stranded RNA (dsRNA) as a gene silencing mechanism has emerged as a next-generation strategy for mite control. RESULTS: We explored the impact of a dsRNA biopesticide, named vadescana, designed to silence the calmodulin gene in Varroa, on mite fitness in mini-hives housed in a laboratory. Two dosages were tested: 2 g/L dsRNA and 8 g/L dsRNA. Vadescana appeared to have no effect on mite survival, however, mite fertility was substantially reduced. The majority of foundress mites exposed to vadescana failed to produce any offspring. No dose-dependent effect of vadescana was observed, as both the low and high doses inhibited mite reproduction equally well in the mini-hives and neither dose impacted pupal survival of the honey bee. Approximately 95% of bee pupae were alive at uncapping across all treatment groups. CONCLUSION: These findings suggest that vadescana has significant potential as an effective alternative to conventional methods for Varroa control, with broader implications for the utilization of RNAi as a next-generation tool in the management of pest species. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Uptake and Biotransformation of Guvermectin in Three Crops after In Vivo and In Vitro Exposure.

Guvermectin, as a novel nucleoside-like biopesticide, could increase the rice yield excellently, but the potential environmental behaviors remain unclear, which pose potential health risks. Therefore, the uptake and biotransformation of guvermectin in three types of crops (rice, lettuce, and carrot) were first evaluated with a hydroponic system. Guvermectin could be rapidly absorbed and reached equilibrium in roots (12-36 h) and shoots (24-60 h) in three plants, and guvermectin was also vulnerable to dissipation in roots (t1/2 1.02-3.65 h) and shoots (t1/2 9.30-17.91 h). In addition, 8 phase I and 2 phase II metabolites, transformed from guvermectin degradation in vivo and in vitro exposure, were identified, and one was confirmed as psicofuranine, which had antibacterial and antitumor properties; other metabolites were nucleoside-like chemicals. Molecular simulation and quantitative polymerase chain reaction further demonstrated that guvermectin was metabolized by the catabolism pathway of an endogenous nucleotide. Guvermectin had similar metabolites in three plants, but the biotransformation ability had a strong species dependence. In addition, all the metabolites exhibit neglectable toxicities (bioconcentration factor <2000 L/kg b.w., LC50,rat > 5000 mg/kg b.w.) by prediction. The study provided valuable evidence for the application of guvermectin and a better understanding of the biological behavior of nucleoside-like pesticides.

Proactive resistance management for sustaining the efficacy of RNA interference for pest control.

Biopesticides based on RNA interference (RNAi) took a major step forward with the first registration of a sprayable RNAi product, which targets the world's most damaging potato pest. Proactive resistance management is needed to delay the evolution of resistance by pests and sustain the efficacy of RNAi biopesticides.

Efficacy and residual toxicity of chitosan for Rhagoletis pomonella (Diptera: Tephritidae).

Chitosan is a naturally derived polymer that has significant potential for use as a bioinsecticide. Despite this, there is a lack of research as to the efficacy of chitosan for many insect pest species. The apple maggot fly, Rhagoletis pomonella (Walsh) (Diptera: Tephritidae), is one such pest for which chitosan toxicity has not been explored. In this study, the toxicity of chitosan for R. pomonella adults was tested via no-choice feeding assays. An aging trial was further used to test the mortality of flies provided dried chitosan-sucrose treatments (CST), which were aged for 0 or 3 days in greenhouse conditions. This study found that the CST is toxic for R. pomonella adults when ingested, leading to a significant increase in the rate of mortality compared to control groups. The use of dried chitosan, however, did not change the mortality of flies, suggesting this will not be an effective delivery mechanism. Effective biopesticide delivery systems have not been defined for chitosan use outside of a laboratory, indicating the need for further research testing delivery mechanisms. It is suggested that an effective method of delivery can be as a food-based bait in attract-and-kill traps, as chitosan must be ingested to kill flies. Forming a viscous solution, chitosan may be best suited to these lure systems.

From weeds to natural enemies: implications of weed cultivation and biopesticides for organic onion production.

Weed management is challenging for vegetable crops that are highly sensitive to weed competition, such as onions. Thrips (Thysanoptera: Thripidae) are major insect pests of onions, causing damage through feeding, and vectoring bacterial pathogens causing bulb rot. Both thrips and their associated pathogens are known to survive on many weed species in onion growing regions. Combining weeding with biopesticides may synergistically manage thrips and reduce disease prevalence. However, disturbances from weeding may negatively impact natural enemies. We estimated the effects of organic weed management and biopesticides on weed density, thrips and natural enemy activity, disease severity, and yield. The experiment was a randomized complete block design, with 4 replications of each weeding (control, tine-weeded twice, tine-weeded 4 times, and hand-weeded) and biopesticide (control, OxiDate 2.0, Serenade) combination. Arthropods were monitored using yellow sticky cards, and weed counts, marketable yield, and bulb rot prevalence were estimated. Hand-weeding resulted in the lowest weed density and thrips abundance. Additionally, hand-weeding produced a 9× higher yield compared to all other treatments. Significant interactions were observed between tine-weeding and biopesticide treatments on the prevalence of bulb rot. Natural enemy abundance was slightly negatively impacted by weeding, dependent on the year. DNA metabarcoding results showed high parasitoid diversity in this onion system and high numbers of reads for multiple genera containing important known biological control agents. Our study suggests hand-weeding is necessary in the southeast for maximum onion yield. Future research should focus on exploring the impact of management on natural enemy communities in onion systems on a large scale.

Biological activity of the essential oil of Drimys winteri.

In the Chilean indigenous culture, the tree Drimys winteri (Winteraceae) Canelo is of great importance and is considered the sacred Mapuche tree. It has antibacterial and disinfectant properties and is used in the treatment of various diseases, such as fevers, ulcers, cancers, and respiratory tract problems. The essential oil obtained from D. winteri, DW_EO, is bioactive, possesses insecticidal and repellent properties against pests, and shows activity toward plant growth regulators. It also has a phytotoxic effect against the growth and germination of weeds. The essential oil obtained from the leaves and bark of Drimys winteri has demonstrated antifungal, immunomodulatory, anti-inflammatory, and anticancer properties in in vitro and in vivo studies. It also possesses antioxidant activity and antibacterial effects. The essential oil contains monoterpenes such as zafrol, pinenes, and linalool, among others, that contribute to its bioactivity. The DW_EO and bioactive compounds have great potential in various applications in medicine, industrial food, sanitizer, and other areas.

Exploring the chemical characterization and insecticidal activities of Curcuma angustifolia roxb. leaf essential oils against three major stored product insects.

Botanical pesticides are safe and widely used in pest management. Curcuma angustifolia belongs to the family Zingiberaceae and is a rhizomatous medicinal herb. Following rhizome harvesting, leaves are discarded as waste. However, they can be effectively utilized by extracting essential oils, which are potential biopesticides. The aim of the study is to evaluate the efficacy of the leaf essential oil of Curcuma angustifolia as a potential biopesticide against three stored grain pests, Lasioderma serricorne, Tribolium castaneum, and Callasobruchus chinensis, by their contact, fumigant, and repellent activities. The leaves yield 0.39 ± 0.02 % of oil by hydrodistillation. GC-MS/MS characterization identified curzerenone (18.37 %), geranyl-p-cymene (17.32 %), α-elemenone (13.59 %), eucalyptol (7.58 %) as the main constituents. When exposed to different concentrations of C. angustifolia oil, the test insect displayed noticeably high repellency rates. It also showed better contact toxicity at 24 h, LC50 = 0.22 mg/cm2 for cigarette beetle, LC50 = 0.64 mg/cm2 for red flour beetle, LC50 = 0.07 mg/cm2 for pulse beetle) and fumigation toxicities (LC50 = 10.8 mg/L air at 24 h, for cigarette, LC50 = 29.5 mg/L air for red flour beetle, LC50 = 7.9 mg/L air for pulse beetle). Additionally, a phytotoxicity study was done on paddy seeds, and the results showed no effect on seed germination or seedling growth. It was evident from this study that C. angustifolia oil from waste leaves can be utilized as a botanical pesticide to manage the adults of these storage pests.

Ben-JNK signaling is required for host mortality during Periplaneta fuliginosa densovirus infection.

BACKGROUND: Cockroaches are widely acknowledged as significant vectors of pathogenic microorganisms. The Periplaneta fuliginosa densovirus (PfDNV) infects the smoky-brown cockroach P. fuliginosa and causes host mortality, which identifies the PfDNV as a species-specific and environmentally friendly biopesticide. However, although the biochemical characterization of PfDNV has been extensively studied, the immune response against PfDNV remains largely unclear. RESULTS: Here, we investigated the replication of PfDNV and its associated pathological phenotype in the foregut and hindgut. Consequently, we dissected and performed transcriptome sequencing on the foregut, midgut, and hindgut separately. We revealed the up-regulation of immune response signaling pathway c-Jun N-terminal kinase (JNK) and apoptosis in response to viral infection. Furthermore, knockdown of the JNK upstream gene Ben resulted in a decrease in virus titer and delayed host mortality. CONCLUSION: Taken together, our findings provide evidence that the Ben-JNK signaling plays a crucial role in PfDNV infection, leading to excessive apoptosis in intestinal tissues and ultimately resulting in the death of the host. Our results indicated that the host response to PfDNV fosters viral infection, thereby increasing host lethality. This underscores the potential of PfDNV as a viable, environmentally friendly biopesticide. © 2024 Society of Chemical Industry.

Plant-derived artificial miRNA effectively reduced the proliferation of aphid (Aphidoidea) through spray-induced gene silencing.

BACKGROUND: Aphids (Hemiptera: Aphididae) are notorious sap-sucking insects that rampantly threaten agricultural production worldwide. Current management against aphids in the field heavily relies on chemical pesticides, which makes economical and eco-friendly methods urgently needed. Spray-induced gene silencing (SIGS) offers a powerful and precise approach to pest management. However, the high costs and instability of double-stranded RNA (dsRNA) regulators applied for downstream RNA interference (RNAi) still limit this strategy. It remains uncertain if RNAi regulators applied in SIGS could extend to small RNA (sRNA), especially miRNA. RESULTS: We chose two sRNA sequences, miR-9b and miR-VgR, whose corresponding targets ABCG4 and VgR are both essential for aphid growth and development. The efficacy of these sequences was initially verified by chemically synthetic single-stranded RNA (syn-ssRNA). Through spray treatment, we observed a significantly decreased survival number and increased abnormality rate of green peach aphids fed on the host under laboratory conditions. Based on our previous study, we generated transgenic plants expressing artificial miR-9b (amiR-9b) and miR-VgR (amiR-VgR). Remarkably, plant-derived amiRNA exerted potent and long-lasting inhibitory efficacy with merely one percent concentration of chemical synthetics. Notably, the simultaneous application of amiR-9b and amiR-VgR exhibited superior inhibitory efficacy. CONCLUSION: We explored the potential use of sRNA-based biopesticide through SIGS while investigating the dosage requirements. To optimize this strategy, the utilization of plant-derived amiRNA was proposed. The results suggested that attributed to stability and durability, deploying amiRNA in pest management is a potential and promising solution for the field application. © 2024 Society of Chemical Industry.

The development of an egg-soaking method for delivering dsRNAs into spider mites.

Recently, the first sprayable RNAi biopesticide, Ledprona, against the Colorado potato beetle, Leptinotarsa decemlineata, has been registered at the United States Environmental Protection Agency. Spider mites (Acari: Tetranychidae), a group of destructive agricultural and horticultural pests, are notorious for rapid development of insecticide/acaricide resistance. The management options, on the other hand, are extremely limited. RNAi-based biopesticides offer a promising control alternative to address this emerging issue. In this study, we i) developed an egg-soaking dsRNA delivery method; ii) evaluated the factors influencing RNAi efficiency, and finally iii) investigated the potential mode of entry of this newly developed egg-soaking RNAi method. In comparison to other dsRNA delivery methods, egg-soaking method was the most efficient, convenient/practical, and cost-effective method for delivering dsRNAs into spider mites. RNAi efficiency of this RNAi method was affected by target genes, dsRNA concentration, developmental stages, and mite species. In general, the hawthorn spider mite, Amphitetranychus viennensis, is more sensitive to RNAi than the two-spotted spider mite, Tetranychus urticae, and both of them have dose-dependent RNAi effect. For different life stages, egg and larvae are the most sensitive life stages to dsRNAs. For different target genes, there is no apparent association between the suppression level and the resultant phenotype. Finally, we demonstrated that this egg-soaking RNAi method acts as both stomach and contact toxicity. Our combined results demonstrate the effectiveness of a topically applied dsRNA delivery method, and the potential of a spray induced gene silencing (SIGS) method as a control alternative for spider mites.

Microsclerotia from Metarhizium robertsii: Production, ultrastructural analysis, robustness, and insecticidal activity.

Microsclerotia (MS) are considered one of the most promising propagules for use as active ingredients in biopesticides due to their tolerance to abiotic factors and ability to produce infective conidia for the control of pests. Therefore, the objective of this research was to establish the conditions required to induce the formation of microsclerotia in Metarhizium robertsii Mt004 and to study its development process, tolerance to abiotic factors and insecticidal activity of MS-derived conidia. M. robertsii started to form hyphal aggregates after 2 days and looked more compact after 8 days. MS were mature and pigmented after 20 days. The final yield was 2.0 × 103 MS/mL and MS size varied between 356.9 and 1348.4 µm. Ultrastructure analysis revealed that mature MS contained only a few live cells embedded in an extracellular matrix. Mature MS were more tolerance to UV-B radiation, heat and storage trials than conidia from Solid State Fermentation. MS-derived conidia were as virulent as conidia against Diatraea saccharalis larvae. These results showed that MS are promising propagules for the development of more persistent and efficient biopesticides for harsh environmental conditions. Our findings provide a baseline for production and a better understanding of microsclerotia development in M. robertsii strains.

N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti.

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58 kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58 kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.