Developing Genetic Engineering Techniques for Control of Seed Size and Yield.

Many signaling pathways regulate seed size through the development of endosperm and maternal tissues, which ultimately results in a range of variations in seed size or weight. Seed size can be determined through the development of zygotic tissues (endosperm and embryo) and maternal ovules. In addition, in some species such as rice, seed size is largely determined by husk growth. Transcription regulator factors are responsible for enhancing cell growth in the maternal ovule, resulting in seed growth. Phytohormones induce significant effects on entire features of growth and development of plants and also regulate seed size. Moreover, the vegetative parts are the major source of nutrients, including the majority of carbon and nitrogen-containing molecules for the reproductive part to control seed size. There is a need to increase the size of seeds without affecting the number of seeds in plants through conventional breeding programs to improve grain yield. In the past decades, many important genetic factors affecting seed size and yield have been identified and studied. These important factors constitute dynamic regulatory networks governing the seed size in response to environmental stimuli. In this review, we summarized recent advances regarding the molecular factors regulating seed size in Arabidopsis and other crops, followed by discussions on strategies to comprehend crops' genetic and molecular aspects in balancing seed size and yield.

For: Pesticide biochemistry and physiology recG is involved with the resistance of Bt to UV.

As an ATP-dependent DNA helicase, RecG can repair DNA replication forks in many organisms. However, knowledge of recG in Bacillus thuringiensis (Bt) is limited. In our previous study, recG was found damaged in Bt LLP29-M19, which was more resistant to ultraviolet light (UV) after exposing Bt LLP29 to UV for 19 generations. To further understand the function of recG in the mechanism of Bt UV resistance, recG was knocked out and recovered with homologous recombination technology in Bt LLP29. Comparing the resistance of the different mutants to UVB, Bt ∆recG-LLP29 lacking recG was found more sensitive to UVB, hydroxyurea (HU) and H2O2 than LLP29 and the complementation strain. To compare the expression level of recG in the Bt strains under different UV treatments, Quantitative Real-time PCR (RT-qPCR) of recG was performed in the tested Bt strains, which showed that the expression level of recG in Bt ∆recG-LLP29 was substantially lower than that in the original strain and complementation strain. Interestingly, when exposed to UV for 20 min, RecG expression in both Bt LLP29 and Bt recG-R was the highest. The unwinding activity of recG in Bt LLP29 and the complementation strain were also found higher than that of the recG knockout strain, Bt ∆recG-LLP29. These results demonstrate that recG is involved with the resistance of Bt to UV. These findings not only enhance the understanding of the Bt UV resistance mechanism, but also provide an important theoretical basis for the application of Bt.

Function of Aedes aegypti galectin-6 in modulation of Cry11Aa toxicity.

Galectins are a family of ß-galactoside binding proteins, and insect galectins play a role in immune responses and may also affect Cry toxin activity. In this study, we aimed to further understand the function and molecular mechanism of Aedes aegypti galectin-6 in modulation of Cry11Aa toxicity. A. aegypti galectin-6 was cloned, and the recombinant galectin-6 was expressed and purified. Bioassays indicated that galectin-6 could reduce the toxicity of Cry11Aa, protecting A. aegypti larvae. To determine interactions among galectin-6, Cry11Aa and putative toxin receptors, Octet Red System, western blotting, far-western blotting and ELISA assays were performed. Octet Red System showed that galectin-6 bound to BBMVs of A. aegypti larvae with lower affinity than that of Cry11Aa. Western blotting and far-western blotting analyses demonstrated that galectin-6 bound to A. aegypti ALP1 and APN2 as well as to BBMVs, consistent with the results of ELISA and protein docking simulations. However, galectin-6 did not bind to Cadherin in far-western blotting or ELISA assay, though the protein docking simulations suggested their binding potential. These findings support the conclusion that galectin-6 may block Cry11Aa from binding to ALP1 and APN2 due to structural similarity, which might decrease the mosquitocidal toxicity of Cry11Aa.

Genome wide survey, evolution and expression analysis of PHD finger genes reveal their diverse roles during the development and abiotic stress responses in Brassica rapa L.

BACKGROUND: Plant homeodomain (PHD) finger proteins are widely present in all eukaryotes and play important roles in chromatin remodeling and transcriptional regulation. The PHD finger can specifically bind a number of histone modifications as an "epigenome reader", and mediate the activation or repression of underlying genes. Many PHD finger genes have been characterized in animals, but only few studies were conducted on plant PHD finger genes to this day. Brassica rapa (AA, 2n = 20) is an economically important vegetal, oilseed and fodder crop, and also a good model crop for functional and evolutionary studies of important gene families among Brassica species due to its close relationship to Arabidopsis thaliana. RESULTS: We identified a total of 145 putative PHD finger proteins containing 233 PHD domains from the current version of B. rapa genome database. Gene ontology analysis showed that 67.7% of them were predicted to be located in nucleus, and 91.3% were predicted to be involved in protein binding activity. Phylogenetic, gene structure, and additional domain analyses clustered them into different groups and subgroups, reflecting their diverse functional roles during plant growth and development. Chromosomal location analysis showed that they were unevenly distributed on the 10 B. rapa chromosomes. Expression analysis from RNA-Seq data showed that 55.7% of them were constitutively expressed in all the tested tissues or organs with relatively higher expression levels reflecting their important housekeeping roles in plant growth and development, while several other members were identified as preferentially expressed in specific tissues or organs. Expression analysis of a subset of 18 B. rapa PHD finger genes under drought and salt stresses showed that all these tested members were responsive to the two abiotic stress treatments. CONCLUSIONS: Our results reveal that the PHD finger genes play diverse roles in plant growth and development, and can serve as a source of candidate genes for genetic engineering and improvement of Brassica crops against abiotic stresses. This study provides valuable information and lays the foundation for further functional determination of PHD finger genes across the Brassica species.

Achromobacter pneumonia in a patient with advanced COPD, a diagnostic challenge.

Bacterial pneumonia causes significant morbidity and mortality especially in elderly and immunocompromised hosts. Achromobacter xylosoxidans denitrificans pneumonia is very rarely reported. However, the reported cases have been in patients who are either immunocompromised or have bronchiectasis. We hereby present a unique case of Achromobacter xylosoxidans denitrificans pneumonia in an immunocompetent patient with advanced chronic obstructive pulmonary disease (COPD). Our patient is a Caucasian male admitted with shortness of breath, fever and cough. Chest X-ray demonstrated right-sided infiltrates and he was treated with intravenous ceftriaxone and azithromycin. He was discharged home on oral amoxicillin-clavulanate 875-125 mg two times per day for a total of 7 days. Patient returned to emergency room after 5 weeks with persistent symptoms and chest X-ray revealed persistent right-sided infiltrate and sputum culture showed Achromobacter xylosoxidans denitrificans. The patient was started on oral levofloxacin 750 mg one time per day for 2 weeks with resolution of symptoms.

Bioengineering Techniques to Improve Nitrogen Transformation and Utilization: Implications for Nitrogen Use Efficiency and Future Sustainable Crop Production.

Nitrogen (N) is crucial for plant growth and development, especially in physiological and biochemical processes such as component of different proteins, enzymes, nucleic acids, and plant growth regulators. Six categories, such as transporters, nitrate absorption, signal molecules, amino acid biosynthesis, transcription factors, and miscellaneous genes, broadly encompass the genes regulating NUE in various cereal crops. Herein, we outline detailed research on bioengineering modifications of N metabolism to improve the different crop yields and biomass. We emphasize effective and precise molecular approaches and technologies, including N transporters, transgenics, omics, etc., which are opening up fascinating opportunities for a complete analysis of the molecular elements that contribute to NUE. Moreover, the detection of various types of N compounds and associated signaling pathways within plant organs have been discussed. Finally, we highlight the broader impacts of increasing NUE in crops, crucial for better agricultural yield and in the greater context of global climate change.

Anaphylactic Shock as a Rare Side Effect of Intravenous Amiodarone.

Amiodarone is a very commonly used antiarrhythmic agent. However, it has a wide variety of systemic side effects as well as many hypersensitivity and allergic reactions, ranging from angioedema to anaphylactic shock in patients who have iodine allergies. We present a rare and unique case of an 86-year-old female who developed anaphylactic shock from intravenous (IV) amiodarone. She had no reported allergies to iodine or iodinated contrast. She had a history of chronic persistent atrial fibrillation and was being maintained on oral amiodarone as an outpatient. She was admitted with shortness of breath and was found to have atrial fibrillation with rapid ventricular response. She was started on an IV amiodarone bolus. Immediately after a few milliliters of infusion, she complained of shortness of breath, with facial flushing and generalized blanching erythema, followed by severe hypotension and cardiopulmonary arrest. IV amiodarone infusion was suspected to be the culprit and was discontinued immediately. IV epinephrine 0.3 mg was administered, followed by the advanced cardiovascular life support (ACLS) protocol for cardiopulmonary arrest. She did not respond to the standard ACLS protocol and continued to remain in cardiopulmonary arrest. A spot diagnosis of anaphylactic reaction to IV amiodarone was made, and she was started on IV epinephrine infusion 0.1 µg/kg/minute, and immediate return of spontaneous circulation was achieved. She was started on IV methylprednisolone 125 mg, IV famotidine 20 mg, and IV diphenhydramine 25 mg. She was intubated and required mechanical ventilation. She was successfully extubated later and safely discharged, receiving oral metoprolol 25 mg for rate control and PO rivaroxaban 20 mg once daily. Anaphylactic shock from IV amiodarone administration is a potentially fatal complication observed in patients with prior reported allergies to iodine or iodinated contrast media. It has rarely been reported in the absence of prior allergy to iodine or iodinated contrast media. Prompt recognition by clinicians is prudent for early diagnosis and appropriate treatment.

Function of CTLGA9 Amino Acid Residue Leucine-6 in Modulating Cry Toxicity.

Aedes aegypti is a crucial vector for many arboviral diseases that cause millions of deaths worldwide and thus is of major public health concern. Crystal (Cry) proteins, which are toxins produced by Bacillus thuringiensis, are structurally organized into three-domains, of which domain II is the most variable in terms of binding towards various toxin receptors. The binding of Cry11Aa to putative receptor such as aminopeptidase-N (APN) is explicitly inhibited by midgut C-type lectins (CTLs). The similarity between the domain II fold of Cry11Aa toxin and the carbohydrate recognition domain in the CTLs is a possible structural basis for the involvement of Cry domain II in the recognition of carbohydrates on toxin receptors. In this study, a site-directed point mutation was introduced into the A. aegypti CTLGA9 gene on the basis of molecular docking findings, leading to substitution of the Leucine-6 (Leu-6) residue in the protein with alanine. Subsequently, functional monitoring of the mutated protein was carried out. Unlike the amino acid residues of wild-type CTLGA9, none of the residues of mutant (m) CTLGA9 were competed with Cry11Aa for binding to the APN receptor interface. Additionally, ligand blot analysis showed that both wild-type and mutant CTLGA9 had similar abilities to bind to APN and Cry11Aa. Furthermore, in the competitive ELISA in which labeled mutant CTLGA9 (10 nM) was mixed with increasing concentrations of unlabeled Cry11Aa (0-500 nM), the mutant showed no competition with Cry11Aa for binding to APN., By contrast, in the positive control sample of labeled wild type CTLGA9 mixed with same concentrations of Cry11Aa competition between the two ligands for binding to the APN was evident. These results suggest that Leucine-6 may be the key site involved in the competitive receptor binding between CTLGA9 and Cry11Aa. Moreover, according to the bioassay results, mutant CTLGA9 could in fact enhance the toxicity of Cry11Aa. Our novel findings provide further insights into the mechanism of Cry toxicity as well as a theoretical basis for enhancing the mosquitocidal activity of these toxin through molecular modification strategies.

Knowledge, attitude, and practices associated with avian influenza among undergraduate university students of East Java Indonesia: A cross-sectional survey.

Background: Several public health strategic actions are required for effective avian influenza (AI) prevention and control, as well as the development of a communication plan to keep undergraduate students sufficiently informed on how to avoid or reduce exposure. The aim of the survey was to measure the level of knowledge, attitudes and practices (KAPs) toward AI among undergraduate university students in East Java, Indonesia, and observe the correlation between KAPs and the factors associated with the control and prevention of AI.  Methods: A cross-sectional survey was conducted among undergraduate students to collect information about AI-related KAPs. Students were selected from three faculties of Universitas Airlangga Surabaya Indonesia (Faculty of Veterinary Medicine, Faculty of Fisheries and Marine, and Faculty of Science and Technology). Students voluntarily responded to a pre-designed questionnaire. Results: A total of 425 students (222 female; and 203 male), of ages ranging from 18 years (n=240) to 20-30 years (n=185), responded to the survey. This cohort consisted of 157 students from the Faculty of Fisheries and Marine, 149 from the Faculty of Veterinary Medicine, and 119 from the Faculty of Science and Technology. The results indicated that appropriate knowledge was obtained by 76.94% of students; significantly higher levels were seen in Faculty of Veterinary Medicine students as compared to the other two faculties (p<0.05). 72.89% of students documented positive attitudes; veterinary medicine students had significantly more positive attitudes than other faculties (p<0.05). Proactive behaviors were observed in 56.90% of students. The aggregate scores for KAPs were 6.93 ± 0.77 (range: 0-9) for knowledge, 7.6 ± 1.25 (range: 0-10) for attitude, and 9.1 ± 1.5 (range: 0-12) for practice.

Role of Lectin in the Response of Aedes aegypti Against Bt Toxin.

Aedes aegypti is one of the world's most dangerous mosquitoes, and a vector of diseases such as dengue fever, chikungunya virus, yellow fever, and Zika virus disease. Currently, a major global challenge is the scarcity of antiviral medicine and vaccine for arboviruses. Bacillus thuringiensis var israelensis (Bti) toxins are used as biological mosquito control agents. Endotoxins, including Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, and Cyt1Aa, are toxic to mosquitoes. Insect eradication by Cry toxin relies primarily on the interaction of cry toxins with key toxin receptors, such as aminopeptidase (APN), alkaline phosphatase (ALP), cadherin (CAD), and ATP-binding cassette transporters. The carbohydrate recognition domains (CRDs) of lectins and domains II and III of Cry toxins share similar structural folds, suggesting that midgut proteins, such as C-type lectins (CTLs), may interfere with interactions among Cry toxins and receptors by binding to both and alter Cry toxicity. In the present review, we summarize the functional role of C-type lectins in Ae. aegypti mosquitoes and the mechanism underlying the alteration of Cry toxin activity by CTLs. Furthermore, we outline future research directions on elucidating the Bti resistance mechanism. This study provides a basis for understanding Bti resistance, which can be used to develop novel insecticides.

Emerging threats and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review.

Avian influenza virus subtype H9N2 was first documented in Indonesia in 2017. It has become prevalent in chickens in many provinces of Indonesia as a result of reassortment in live bird markets. Low pathogenic avian influenza subtype H9N2 virus-infected poultry provides a new direction for influenza virus. According to the latest research, the Indonesian H9N2 viruses may have developed through antigenic drift into new genotype, posing a significant hazard to poultry and public health. The latest proof of interspecies transmission proposes that, the next human pandemic variant will be avian influenza virus subtype H9N2. Manipulation and elimination of H9N2 viruses in Indonesia, constant surveillance of viral mutation, and vaccines updates are required to achieve effectiveness. The current review examines should be investigates/assesses/report on the development and evolution of newly identified H9N2 viruses in Indonesia and their vaccination strategy.

Densovirus Oil Suspension Significantly Improves the Efficacy and Duration of Larvicidal Activity against Aedes albopictus.

Aedes albopictus is the sole vector for various mosquito-borne viruses, including dengue, chikungunya, and Zika. Ecofriendly biological agents are required to reduce the spread of these mosquito-borne infections. Mosquito densoviruses (MDVs) are entomopathogenic mosquito-specific viruses, which can reduce the capacity of isolated vectors and decrease mosquito-borne viral disease transmission. However, their variable pathogenicity restricts their commercial use. In the present study, we developed a series of novel larvicide oil suspensions (denoted Bacillus thuringiensis (Bti) oil, Ae. albopictus densovirus (AalDV-5) oil, and a mixture of AalDV-5+Bti oil), which were tested against Ae. albopictus larvae under experimental semi-field and open-field conditions. The effect of AalDV-5 on non-target species was also evaluated. The combined effect of AalDV-5+Bti was greater than that of individual toxins and was longer lasting and more persistent compared with the laboratory AalDV-5 virus strain. The virus was quantified on a weekly basis by quantitative polymerase chain reaction (qPCR) and was persistently detected in rearing water as well as in dead larvae. Wildtype densovirus is not pathogenic to non-target organisms. The present findings confirm the improved effect of a mixed microbial suspension (AalDV-5+Bti oil) larvicide against Ae. albopictus. The development and testing of these products will enable better control of the vector mosquitoes.

Recombinant Mosquito Densovirus with Bti Toxins Significantly Improves Pathogenicity against Aedes albopictus.

Mosquito densoviruses (MDVs) are mosquito-specific viruses that are recommended as mosquito bio-control agents. The MDV Aedes aegypti densovirus (AeDNV) is a good candidate for controlling mosquitoes. However, the slow activity restricts their widespread use for vector control. In this study, we introduced the Bacillus thuringiensis (Bti) toxin Cry11Aa domain II loop α8 and Cyt1Aa loop ß6-αE peptides into the AeDNV genome to improve its mosquitocidal efficiency; protein expression was confirmed using nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Recombinant plasmids were transfected into mosquito C6/36 cell lines, and the expression of specific peptides was detected through RT-PCR. A toxicity bioassay against the first instar Aedes albopictus larvae revealed that the pathogenic activity of recombinant AeDNV was significantly higher and faster than the wild-type (wt) viruses, and mortality increased in a dose-dependent manner. The recombinant viruses were genetically stable and displayed growth phenotype and virus proliferation ability, similar to wild-type AeDNV. Our novel results offer further insights by combining two mosquitocidal pathogens to improve viral toxicity for mosquito control.

Comprehensive genomic survey, structural classification and expression analysis of C2H2 zinc finger protein gene family in Brassica rapa L.

C2H2 zinc finger protein (ZFP) genes have been extensively studied in many organisms and can function as transcription factors and be involved in many biological processes including plant growth and development and stress responses. In the current study, a comprehensive genomics analysis of the C2H2-ZFP genes in B. rapa was performed. A total of 301 B. rapa putative C2H2-ZFP (BrC2H2-ZFP) genes were identified from the available Brassica genome databases, and further characterized through analysis of conserved amino acid residues in C2H2-ZF domains and their organization, subcellular localization, phylogeny, additional domain, chromosomal location, synteny relationship, Ka/Ks ratio, and expression pattern. We also analyzed the expression patterns of eight B. rapa C2H2-ZFP genes under salt and drought stress conditions by using qRT-PCR technique. Our results showed that about one-third of these B. rapa C2H2-ZFP genes were originated from segmental duplication caused by the WGT around 13 to 17 MYA, one-third of them were highly and consecutively expressed in all tested tissues, and 92% of them were located in nucleus by prediction supporting then their functional roles as transcription factors, of which some may play important roles in plant growth and development. The Ka/Ks ratios of 264 orthologous C2H2-ZFP gene pairs between A. thaliana and B. rapa were all, except two, inferior to 1 (varied from 0.0116 to 1.4919, with an average value of 0.3082), implying that these genes had mainly experienced purifying selection during species evolution. The estimated divergence times of the same set of gene pairs ranged from 6.23 to 38.60 MY, with an average value of 18.29 MY, indicating that these gene members have undergone different selective pressures resulting in different evolutionary rates during species evolution. In addition, a few of these B. rapa C2H2-ZFPs were shown to be involved in stress responses in a similar way as their orthologs in A. thaliana. Comparison between A. thaliana and B. rapa orthologous C2H2-ZFP genes showed that the majority of these C2H2-ZFP gene members encodes proteins with conserved subcellular localization and functional domains between the two species but differed in their expression patterns in five tissues or organs. Thus, our study provides valuable information for further functional determination of each C2H2-ZFP gene across the Brassica species, and may help to select the appropriate gene targets for further in-depth studies, and genetic engineering and improvement of Brassica crops.

Comprehensive Genomic Survey, Characterization and Expression Analysis of the HECT Gene Family in Brassica rapa L. and Brassica oleracea L.

The HECT-domain protein family is one of the most important classes of E3 ligases. While the roles of this family in human diseases have been intensively studied, the information for plant HECTs is limited. In the present study, we performed the identification of HECT genes in Brassica rapa and Brassica oleracea, followed by analysis of phylogeny, gene structure, additional domains, putative cis-regulatory elements, chromosomal location, synteny, and expression. Ten and 13 HECT genes were respectively identified in B. rapa and B. oleracea and then resolved into seven groups along with their Arabidopsis orthologs by phylogenetic analysis. This classification is well supported by analyses of gene structure, motif composition within the HECT domain and additional protein domains. Ka/Ks ratio analysis showed that these HECT genes primarily underwent purifying selection with varied selection pressures resulting in different rates of evolution. RNA-Seq data analysis showed that the overwhelming majority of them were constitutively expressed in all tested tissues. qRT-PCR based expression analysis of the 10 B. rapa HECT genes under salt and drought stress conditions showed that all of them were responsive to the two stress treatments, which was consistent with their promoter sequence analysis revealing the presence of an important number of phytohormone-responsive and stress-related cis-regulatory elements. Our study provides useful information and lays the foundation for further functional determination of each HECT gene across the Brassica species.

CTLGA9 Interacts with ALP1 and APN Receptors To Modulate Cry11Aa Toxicity in Aedes aegypti.

The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans, including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). Bacillus thuringiensis (Bt) is widely used as a biopesticide, which produces Cry toxins for mosquito control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in response to Cry toxins. Our results showed by far-western blot and ELISA methods that the CTLTGA9 protein interacted with brush border membrane vesicles (BBMVs) of A. aegypti larvae and with ALP1, APN, and Cry11Aa proteins. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the larvicidal activity of Cry toxins. According to the results of molecular docking, CTLGA9 may compete with Cry11Aa for binding to ALP1 and APN receptors and thus decreases the mosquitocidal toxicity of Cry11Aa. Our results provide further insights into better understanding the mechanism of Cry toxins and help improve the Cry toxicity for mosquito control.

Cloning, expression and activity of ATP-binding protein in Bacillus thuringiensis toxicity modulation against Aedes aegypti.

BACKGROUND: Bacillus thuringiensis israelensis (Bti) is a widely used mosquitocidal microbial pesticide due to its high toxicity. ATP-binding proteins (ABP) are prevalently detected in insects and are related to reaction against Bti toxins. However, the function of ABP in mosquito biocontrol is little known, especially in Aedes aegypti. Therefore, this study aimed to clarify the function of ABP in Ae. aegypti against Bti toxin. RESULTS: Aedes aegypti ABP (GenBank: XM_001661856.2) was cloned, expressed and purified in this study. Far-western blotting and ELISA were also carried out to confirm the interaction between ABP and Cry11Aa. A bioassay of Cry11Aa was performed both in the presence and absence of ABP, which showed that the mortality of Ae. aegypti is increased with an increase in ABP. CONCLUSIONS: Our results suggest that ABP in Ae. aegypti can modulate the toxicity of Cry11Aa toxin to mosquitoes by binding to Bti toxin. This could not only enrich the mechanism of Bt toxin, but also provide more data for the biocontrol of this transmission vector.

C-Type Lectin-20 Interacts with ALP1 Receptor to Reduce Cry Toxicity in Aedes aegypti.

Aedes aegypti is a crucial vector for human diseases, such as yellow fever, dengue, chikungunya, and Zika viruses. Today, a major challenge throughout the globe is the insufficient availability of antiviral drugs and vaccines against arboviruses, and toxins produced by Bacillus thuringiensis (Bt) are still used as biological agents for mosquito control. The use of Cry toxins to kill insects mainly depends on the interaction between Cry toxins and important toxin receptors, such as alkaline phosphatase (ALP). In this study, we investigated the function of A. aegypti C-type lectin-20 (CTL-20) in the tolerance of Cry toxins. We showed that recombinant CTL-20 protein interacted with both Cry11Aa and ALP1 by the Far-Western blot and ELISA methods, and CTL-20 bound to A. aegypti larval brush border membrane vesicles (BBMVs). Binding affinity of CTL-20 to ALP1 was higher than that of Cry11Aa to ALP1. Furthermore, the survival rate of A. aegypti larvae fed with Cry11Aa toxin mixed with recombinant CTL-20 fusion protein was significantly increased compared with that of the control larvae fed with Cry11Aa mixed with thioredoxin. Our novel results suggest that midgut proteins like CTLs may interfere with interactions between Cry toxins and toxin receptors by binding to both Cry toxins and receptors to alter Cry toxicity.

Transcriptomic Analysis of Aedes aegypti in Response to Mosquitocidal Bacillus thuringiensis LLP29 Toxin.

Globally, Aedes aegypti is one of the most dangerous mosquitoes that plays a crucial role as a vector for human diseases, such as yellow fever, dengue, and chikungunya. To identify (1) transcriptomic basis of midgut (2) key genes that are involved in the toxicity process by a comparative transcriptomic analysis between the control and Bacillus thuringiensis (Bt) toxin (LLP29 proteins)-treated groups. Next-generation sequencing technology was used to sequence the midgut transcriptome of A. aegypti. A total of 17130 unigenes, including 574 new unigenes, were identified containing 16358 (95.49%) unigenes that were functionally annotated. According to differentially expressed gene (DEG) analysis, 557 DEGs were annotated, including 226 upregulated and 231 downregulated unigenes in the Bt toxin-treated group. A total of 442 DEGs were functionally annotated; among these, 33 were specific to multidrug resistance, 6 were immune-system-related (Lectin, Defensin, Lysozyme), 28 were related to putative proteases, 7 were lipase-related, 8 were related to phosphatases, and 30 were related to other transporters. In addition, the relative expression of 28 DEGs was further confirmed through quantitative real time polymerase chain reaction. The results provide a transcriptomic basis for the identification and functional authentication of DEGs in A. aegypti.

Comparison and Mechanism of the UV-Resistant Mosquitocidal Bt Mutant LLP29-M19.

Bacillus thuringiensis (Bt) is one of the most widely used and studied biopesticides. However, it is vulnerable to the influence of ultraviolet (UV) radiation, causing shorter persistence under field conditions. To obtain a high-active and effective Bt new product, the main objective of this study is to obtain a highly UV-resistant Bt mutant from the mosquitocidal Bt LLP29 through UV exposure. After 19 rounds of UV exposure, a Bt mutant named LLP29-M19 was obtained, showing resistance to UV radiation for up to 67 min. The mosquitocidal fatality rate of LLP29-M19 was 95%, which was slightly higher than that of LLP29 (90%). Comparative characterization showed that there were no substantial differences in morphology between LLP29-M19 and the original strain, LLP29. However, some changes were detected in physiological and biochemical characteristic reactions, including fructose, glucose, and xylose metabolism. Furthermore, although both LLP29-M19 and LLP29 showed negative zeta potentials, the surface charge of LLP29 was -28.1 mV and that of LLP29-M19 was -42.8 mV. The size distribution of LLP29-M19 was also slightly larger than that of LLP29. Fourier transform infrared analysis indicated that amide functional groups might be involved in the resistance mechanism of LLP29-M19. Quantitative analysis using inductive coupled plasma emission spectrometry showed that some elements increased greatly in LLP29-M19, such as K. All of these results will be highly valuable for better understanding the mechanism of Bt resistance. Explanations regarding the resistance mechanism of this novel Bt mutant may lead to the development of new biopesticides with high mosquitocidal activity and persistence.