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Abstract Regenerating inflamed bone defects represents a severe clinical challenge due to the undesirable inflammatory microenvironment. The inflammatory stimulus poses a weighty threat to the regenerative capacity of endogenously derived mesenchymal stem cells (MSCs), which are mainly responsible for osteogenic differentiation, thereby resulting in compromised endogenous bone formation. Consequently, alleviating the biological characteristics of inflammatory-impaired MSCs is crucial for promoting inflamed bone regeneration. Nano-sized small extracellular vesicles (sEVs) have emerged as promising therapeutic tools to orchestrate MSCs fate due to their intrinsic biocompatibility and encapsulated bioactive contents. In the present study, we extracted sEVs from youthful and adult dental pulp MSCs and explored their ability to recover inflammation-compromised periodontal ligament stem cells (IPDLSCs). The results indicated that both types of sEVs were capable of facilitating IPDLSCs osteogenesis. However, young sEVs exhibited a more robust potential at a lower concentration compared to adult sEVs. Mechanically, young sEVs enhanced the expression of bone morphogenetic protein 4 (BMP4) via delivering the protein Biglycan, which correspondingly promoted the osteogenic capability of IPDLSCs. Collectively, our findings emphasized that young sEVs hold enormous potential to rescue the inherent function and regenerative competence of inflammation-impaired MSCs, shedding light on their promising therapeutic prospects for infected bone regeneration.

Conogethes punctiferalis is a crop and fruit pest that has caused serious economic losses to agricultural production. This pest relies heavily on its sex pheromone to ensure sexual encounters and subsequent mating success. However, the molecular mechanism underlying sex pheromone biosynthesis in this species remains elusive. The present study investigated the detailed mechanism underlying PBAN-regulated sex pheromone biosynthesis in C. punctiferalis by transcriptome sequencing of the C. punctiferalis pheromone glands (PGs) and subsequent functional identification of the target genes. The results showed that female mating started from the first scotophase, and peaked at the second to fifth scotophases in accordance with the release of sex pheromones. PBAN regulated sex pheromone biosynthesis by employing Ca2+ and cAMP as secondary messengers, as demonstrated by RNA interference (RNAi), pharmacological inhibitors, and behavioral assays. Further investigation revealed that calcineurin (CaN) and acetyl-CoA carboxylase (ACC) were activated by PBAN/Ca2+ signaling, and the RNAi-mediated knockdown of CaN and ACC transcripts significantly reduced sex pheromone production, ultimately leading to a significantly reduced ability of females to attract males. Importantly, hexokinase (HK) was found to regulate sex pheromone biosynthesis in response to the PBAN/cAMP/PKA signaling pathway, as demonstrated by RNAi, enzyme activity, and pharmacological inhibitor assays. Furthermore, Far2 and Desaturase1 were found to participate in PBAN-regulated sex pheromone biosynthesis. Altogether, our findings revealed that PBAN regulates sex pheromone biosynthesis through the PBANR/Ca2+/CaN/ACC and PBANR/cAMP/PKA/HK pathways in C. punctiferalis, which enriches our comprehension of the details of sex pheromone biosynthesis in moths.

Helicoverpa zea is a major target pest of pyramided transgenic crops expressing Cry1, Cry2 and/or Vip3Aa proteins from Bacillus thuringiensis (Bt) in the United States. Laboratory-selected Cry1Ac/Cry2Ab cross resistance and field-evolved practical dual resistance of H. zea to these two toxins have been widely reported. Whether the widespread Cry1Ac/Cy2Ab dual resistance of H. zea has resulted from the selection of one shared or two independent resistance mechanisms by pyramided Bt crops remains unclear. Cadherin is a well-confirmed receptor of Cry1Ac and a suggested receptor of Cry2Ab in at least three Lepidopteran species. To test whether cadherin may serve as one shared mechanism for the cross and dual resistance of H. zea to Cry1Ac and Cry2Ab, we cloned H. zea cadherin (HzCadherin) cDNA and studied its functional roles in the mode of action of Cry1Ac and Cry2Ab by gain- and loss-of-function analyses. Heterologous expression of HzCadherin in H. zea midgut, H. zea fat body and Sf9 cells made all three of these cell lines more susceptible to activated Cry1Ac but not activated Cry2Ab, whereas silencing HzCadherin of H. zea midgut and fat body cells significantly reduced the susceptibility to Cry1Ac but not Cry2Ab. Likewise, suppressing HzCadherin with siRNA made H. zea larvae resistant to Cry1Ac. These results clearly demonstrate that HzCadherin is not a receptor for Cry2Ab, and thus it is unlikely to serve as one shared mechanism for the cross and dual resistance of H. zea to Cry1Ac and Cry2Ab.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis, is commonly used as an internal reference gene in humans, mice, and insects. However, the function of GAPDH in insect development, especially in metamorphosis, has not been reported. In the present study, Helicoverpa armigera and Spodoptera frugiperda ovarian cell lines (Sf9 cells) were used as materials to study the function and molecular mechanism of GAPDH in larval metamorphosis. The results showed that HaGAPDH was more closely related to GAPDH of S. frugiperda and Spodoptera litura. The transcript peaks of HaGAPDH in sixth instar larvae were 6L-3 (epidermal and midgut) and 6L-1 (fat body) days, and 20E and methoprene significantly upregulated the transcripts of HaGAPDH of larvae in qRT-PCR. HaGAPDH–GFP–His was specifically localized in mitochondria in Sf9 cells. Knockdown of HaGAPDH by RNA interference (RNAi) in sixth instar larvae resulted in weight loss, increased mortality, and decreases in the pupation rate and emergence rates. HaGAPDH is directly bound to soluble trehalase (HaTreh1) physically and under 20E treatment in yeast two-hybrid, coimmunoprecipitation, and colocalization experiments. In addition, knockdown of HaGAPDH increased the Treh1 activity, which in turn decreased the trehalose content but increased the glucose content in larvae. Therefore, these data demonstrated that GAPDH controlled the glucose content within the normal range to ensure glucose metabolism and metamorphosis by directly binding with HaTreh1.

Bacillus thuringiensis (Bt) is the safest, economically successful entomopathogen to date. It is extensively produced in transgenic crops or used in spray formulations to control Lepidopteran pests. The most serious threat to the sustainable usage of Bt is insect resistance. The resistance mechanisms to Bt toxins depend not only on alterations in insect receptors, but also on the enhancement of insect immune responses. In this work, we review the current knowledge of the immune response and resistance of insects to Bt formulations and Bt proteins, mainly in Lepidopteran pests. We discuss the pattern recognition proteins for recognizing Bt, antimicrobial peptides (AMPs) and their synthetic signaling pathways, the prophenoloxidase system, reactive oxygen species (ROS) generation, nodulation, encapsulation, phagocytosis, and cell-free aggregates, which are involved in immune response reactions or resistance to Bt. This review also analyzes immune priming, which contributes to the evolution of insect resistance to Bt, and puts forward strategies to improve the insecticidal activity of Bt formulations and manage insect resistance, targeting the insect immune responses and resistance.

Helicoverpa armigera (cotton bollworm) is one of the most destructive pests worldwide. Due to resistance to Bacillus thuringiensis and conventional insecticides, an effective management strategy to control this pest is urgently needed. Spinosad, a natural pesticide, is considered an alternative; however, the mechanism underlying the developmental effects of sublethal spinosad exposure remains elusive. In this study, the mechanism was examined using an insect model of H. armigera. Results confirmed that exposure to sublethal spinosad led to reduced larval wet weight, delayed larval developmental period, caused difficulty in molting, and deformed pupae. Further investigation demonstrated that exposure to sublethal spinosad caused a significant decrease in 20E titer and increase in JH titer, thereby leading to the discordance between 20E and JH titers, and consequently alteration in the expression levels of HR3 and Kr-h1. These results suggested that sublethal spinosad caused hormonal disorders in larvae, which directly affect insect development. Our study serves as a reference and basis for the toxicity evaluation of spinosad on molting and pupation in insect metamorphosis, which may contribute to identifying targets for effective control of cotton bollworm.

Female moths use sex pheromones to attract males, and corresponding regulatory mechanism underlying sex pheromone biosynthesis is species-dependent. However, the detailed mechanism involved in sex pheromone biosynthesis in Ostrinia furnacalis has not yet been fully addressed. In the present study, transcriptome sequencing of O. furnacalis pheromone glands screened a serials of candidate genes involved in sex pheromone biosynthesis. Our analysis showed that sex pheromone release in O. furnacalis females arrives its peak at the 2nd scotophase, consistent with its mating behavior. Pheromone biosynthesis-activating neuropeptide (PBAN) was confirmed to regulate sex pheromone biosynthesis, and Ca2+ is the secondary messenger of PBAN signaling in O. furnacalis. The functional analysis of candidate genes demonstrated that the decreased mRNA levels or activities of calcineurin (CaN) and acetyl-CoA carboxylase (ACC) led to significant decrease in sex pheromone production and female capability to attract males, as demonstrated by RNAi-mediated knockdown and pharmacological inhibitor assay. Most importantly, the activities of CaN and ACC depend on the activation of PBAN/PBANR/Ca2+. Furthermore, fatty-acyl reductase 14 was involved in PBAN-mediated sex pheromone biosynthesis. Altogether, our results demonstrated that PBAN regulates sex pheromone biosynthesis through PBANR/Ca2+/CaN/ACC pathway to promote sex pheromone biosynthesis in O. furnacalis and provided a reference for non-model organism to study neuropeptide signal transduction.

The long-term use of chemical insecticides has caused serious problems of insect resistance and environmental pollution; new insecticides are needed to solve this problem. Cyclosporin A (CsA) is a polypeptide produced by many fungi, which is used to prevent or treat immune rejection during organ transplantation. However, little is known about the utility of CsA as an insecticide. Therefore, this study evaluated the insecticidal activity of CsA using Ostrinia furnacalis as a model. The results demonstrated that CsA was toxic to O. furnacalis with LC50 values of 113.02 μg/g and 198.70 μg/g for newly hatched neonates and newly molted third-instar larvae, respectively. Furthermore, CsA treatment had sublethal effects on the development of O. furnacalis, and significantly reduced the fecundity of adults; this suggests that CsA has great potential to suppress O. furnacalis populations. Further analysis revealed that CsA suppressed calcineurin activity in larvae. CsA had independent or synergistic toxic effects on O. furnacalis when combined with β-cypermethrin, indoxacarb, emamectin benzoate, azadirachtin, and the Bacillus thuringiensis toxin Cry1Ac, which suggests that CsA can help prevent or manage resistance. Our study provides detailed information on the potential of CsA as an insecticide for controlling lepidopterans.

The fall armyworm (FAW), Spodoptera frugiperda, causes substantial annual agricultural production losses worldwide due to its resistance to many insecticides. Therefore, new insecticides are urgently needed to more effectively control FAW. Cyclosporin A (CsA) is a secondary metabolite of fungi; little is known about its insecticidal activity, especially for the control of FAW. In this study, we demonstrate that CsA shows excellent insecticidal activity (LC50 = 9.69 μg/g) against FAW through significant suppression of calcineurin (CaN) activity, which is a new target for pest control. Combinations of CsA and indoxacarb, emamectin benzoate, or Vip3Aa showed independent or synergistic toxicity against FAW; however, the combination of CsA and chlorantraniliprole showed no toxicity. Sublethal doses of CsA led to decreases in FAW larval and pupal weight, pupation, emergence, mating rates, adult longevity, extended development of FAW larvae and pupae and the pre-oviposition period of adults, and increases in the proportion of pupal malformation. Importantly, CsA treatment reduced FAW ovarian size and female fecundity, which suggests that it has great potential to suppress FAW colony formation. Taken together, these results indicate that CsA has high potential as an insecticide for controlling FAW.

Termite soldiers are a unique caste among social insects, and their differentiation can be induced by Juvenile hormone (JH) from workers through two molts (worker–presoldier–soldier). However, the molecular mechanism underlying the worker-to-soldier transformation in termites is poorly understood. To explore the mechanism of soldier differentiation induced by JH, the gene soldier caste-specific protein 1 (RaSsp1, NCBI accession no: MT861054.1) in R. aculabialis was cloned, and its function was studied. This gene was highly expressed in the soldier caste, and the protein RsSsp1 was similar to the JHBP (JH-binding protein) domain-containing protein by Predict Protein online. In addition, JHIII could be anchored in the hydrophobic cage of RaSsp1 as the epoxide of the JHBP-bound JH according to the protein ligand molecular docking online tool AutoDock. The functional studies indicated that knocking down of the RaSsp1 shorted the presoldier’s head capsule, reduced mandible size, delayed molting time and decreased molting rate (from worker to presoldier) at the beginning of worker gut-purging. Furthermore, knocking down of the RaSsp1 had a more pronounced effect on soldier differentiation (from presoldier to soldier), and manifested in significantly shorter mandibles, rounder head capsules, and lower molting rate (from worker to presoldier) at the beginning of presoldier gut-purging. Correspondingly, the expressions of JH receptor Methoprene-tolerant (Met), the JH-inducible transcription factor Krüppel homolog1 (Kr-h1) and ecdysone signal genes Broad-complex (Br-C) were downregulated when knocking down the RaSsp1 at the above two stages. All these results that RaSsp1 may be involved in soldier differentiation from workers by binding and transporting JH.

Insect resistance to Bacillus thuringiensis (Bt) insecticidal proteins has rapidly evolved with the expansion of the planting area of transgenic Bt crops. Pyramiding RNA interference (RNAi) and Bt in crops is urgently needed to counter the rapid increase in pest resistance. The ideal “pyramid” strategy simultaneously targets different action pathways that exert synergetic effects on each other. Here, we identified a dephosphatase, namely, Helicoverpa armigera calcineurin (HaCAN), which might enhance the insecticidal activity of Cry1Ac against Helicoverpa armigera by regulating immune gene expression via dephosphatase activity, but not by acting as a receptor. Notably, blocking enzyme activity or knocking down endogenous HaCAN significantly promoted the enhancement in Cry1Ac toxicity to insect larvae and cells. Correspondingly, the increase in HaCAN activity reduced the cytotoxicity of Cry1Ac as shown by the heterologous expression of HaCAN. Our results provide a probable that HaCAN is an important candidate gene for pyramiding RNAi and Cry1Ac crops to control cotton bollworm.

Cell culture is one of the most core and fundamental techniques employed in the fields of biology and medicine. At present, although the two-dimensional cell culture method is commonly used in vitro, it is quite different from the cell growth microenvironment in vivo. In recent years, the limitations of two-dimensional culture and the advantages of three-dimensional culture have increasingly attracted more and more attentions. Compared to two-dimensional culture, three-dimensional culture system is better to realistically simulate the local microenvironment of cells, promote the exchange of information among cells and the extracellular matrix (ECM), and retain the original biological characteristics of stem cells. In this review, we first present three-dimensional cell culture methods from two aspects: a scaffold-free culture system and a scaffold-based culture system. The culture method and cell characterizations will be summarized. Then the application of three-dimensional cell culture system is further explored, such as in the fields of drug screening, organoids and assembloids. Finally, the directions for future research of three-dimensional cell culture are stated briefly.

Supplemental nutrients of adult moths maximize moth fitness and contribute to the pollination of many plants. Previous reports have revealed that sugar feeding promotes to sex pheromone biosynthesis by increasing the haemolymph trehalose concentration in mating moths. Here, Mythimna separata adults were employed as a model to investigate the effect of sugar feeding on sex pheromone biosynthesis. Results showed that in virgin females, sugar feeding markedly increased the concentrations of trehalose, pyruvic acid, and acyl-CoA in pheromone glands (PGs), which in turn led to an increase in sex pheromone titer, female ability to attract males and successfully mating frequency in sugar-fed females. Consistently, sugar-fed females laid more eggs than water-fed females. Furthermore, the refeeding of starved females also caused significantly increase in the concentrations of trehalose, pyruvic acid, and acyl-CoA in PGs, thus facilitating a significant increase in sex pheromone production. Most importantly, RNAi-mediated knockdown of trehalase (leading to PG starvation) resulted in an increase in trehalose content, and decrease in the concentrations of pyruvic acid, and acyl-CoA in PGs, which in turn led to a decrease of sex pheromone titer, female ability to attract males and successful mating efficacy. Altogether, results revealed a mechanism by which sugar feeding contributed to trehalose utilization in PGs, promoted to significantly increased sex pheromone precursor by increasing the concentrations of pyruvic acid and acyl-CoA, and facilitated to sex pheromone biosynthesis and successful mating.

Helicoverpa armigera is a universal pest around the world that has been extensively used as a model organism for agricultural pests. Calcineurin (CAN) is an important Ca2+-dependent phosphatase that is participated in various biological pathways. Here, we revealed that CAN inhibition significantly arrested H. armigera larval development by reducing larvae weight, prolonging development time and reducing pupate rates. Furthermore, CAN serves as an immune activator and regulates antimicrobial peptide (AMP; cecropin D, attacin, and gloverin) expression by binding with relish transcript factor in H. armigera. This study provides a potential target to control H. armigera by using synergistic agents for pesticides or plant-mediated RNA interference technology.

Helicoverpa armigera can develop resistance to Bacillus thuringiensis (Bt), which threaten the long-term success of Bt crops. In the present study, RNAseq was employed to investigate the midgut genes response to strains with different levels of resistance (LF5, LF10, LF20, LF30, LF60, and LF120) in H. armigera. Results revealed that a series of differentially expressed unigenes (DEGs) were expressed significantly in resistant strains compared with the LF-susceptible strain. Nine trypsin genes, ALP2, were downregulated significantly in all the six resistant strains and further verified by qRT-PCR, indicating that these genes may be used as markers to monitor and manage pest resistance in transgenic crops. Most importantly, the differences in DEG functions in the different resistant strains revealed that different resistance mechanisms may develop during the evolution of resistance. The immune and detoxification processes appear to be associated with the low-level resistance (LF5 strain). Metabolic process-related macromolecules possibly lead to resistance to Cry1Ac in the LF10 and LF20 strains. The DEGs involved in the “proton-transporting V-type ATPase complex” and the “proton-transporting two-sector ATPase complex” were significantly expressed in the LF30 strain, probably causing resistance to Cry1Ac in the LF30 strain. The DEGs involved in binding and iron ion homeostasis appear to lead to high-level resistance in the LF60 and LF120 strains, respectively. The multiple genes and different pathways seem to be involved in Cry1Ac resistance depending on the levels of resistance. Although the mechanisms of resistance are very complex in H. armigera, a main pathway seemingly exists, which contributes to resistance in each level of resistant strain. Altogether, the findings in the current study provide a transcriptome-based foundation for identifying the functional genes involved in Cry1Ac resistance in H. armigera.

A pyramid strategy combining the crystal (Cry) 1A and 2A toxins in Bacillus thuringiensis (Bt) crops are active against many species of insects and nematode larvae. It has been widely used to delay pest adaption to genetically modified plants and broaden the insecticidal spectrum in many countries. Unfortunately, Cry2A can also bind with the specific receptor proteins of Cry1A. ATP-binding cassette (ABC) transporters can interact with Cry1A toxins as receptors in the insect midgut, and ABC transporter mutations result in resistance to Bt proteins. However, there is limited knowledge of the ABC transporters that specifically bind to Cry2Ab. Here, we cloned the ABCC1 gene in Helicoverpa armigera, which expressed at all larval stages and in nine different tissues. Expression levels were particularly high in fifth-instar larvae and Malpighian tubules. The two heterologously expressed HaABCC1 transmembrane domain peptides could specifically bind to Cry2Ab with high affinity levels. Moreover, transfecting HaABCC1 into the Spodoptera frugiperda nine insect cell significantly increased its mortality when exposed to Cry2Ab in vitro, and silencing HaABCC1 in H. armigera by RNA interference significantly reduced the mortality of larvae exposed to Cry2Ab in vivo. Altogether current results suggest that HaABCC1 serves as a functional receptor for Cry2Ab.

Helicoverpa armigera is an universal pest around the world, which has recovered again in recent years because of the adjustment of cropping structure and resistance to Bacillus thuringiensis (Bt) in China. As a new insecticide spinetoram is extensively used to control many pest insects, including H. armigera. However the lethal and sublethal effects of spinetoram on cotton bollworm have not been assessed. In the present study, the toxicity of spinetoram against cotton bollworm was tested under laboratory conditions. Results demonstrated spinetoram showed an excellent activity against H. armigera, especially, against Bt (Cry1Ac) resistant H. armigera. Treatment with spinetoram at the doses of 0.19 mg/kg and 0.36 mg/kg (LC8 and LC20 after 24h oral exposure) significantly arrested the development of surviving larvae and caused significant decrease in larvae wet weight. Besides, the survivors after spinetoram treatments showed significant reduction of pupation ratio, pupal weight, emergence ratio, longevity and fecundity of adults. At same time, spinetoram treatments resulted in significant increase in the prepupal and pupal periods of survivors. In summary, these results showed that spinetoram could be used as an effective pesticide to control H. armigera, especially Cry1Ac-ressitacne, consequently to take both lethal and sublethal effects to cotton bollworm into consideration in cotton bollworm control strategy.

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Beta-cypermethrin is a commonly used insecticide for controlling major agricultural pests as Helicoverpa armigera in crop fields. This study investigated the sublethal effect of beta-cypermethrin on the mating physiology of H. armigera. The results demonstrated that the sublethal exposure to beta-cypermethrin significantly decreased sex pheromone production, hindered female ability to attract males and reduced the proportion of successful matings. Further investigations revealed that trehalose content in the sex pheromone glands (PGs) significantly decreased following sublethal beta-cypermethrin treatment, leading to a subsequent decrease of acetyl-CoA content within the PGs. Furthermore, such exposure caused a significant decrease in mRNA expression levels of the trehalose transporter, calcineurin (CaN), acetyl CoA carboxylase (ACC), protein kinase A (PKA), pheromone biosynthesis-activating neuropeptide receptor (PBANR), Δ11-desaturase (DES) and fatty acid reductase 2 (FAR2), as well as a decrease in the enzyme activities of membranebound trehalase, ACC, CaN and PKA. Sublethal exposure lowered the expression levels of pheromone binding protein 1, pheromone binding protein 2 and odorant receptor 13 in males, suggesting decreased male recognition capabilities. Altogether, sublethal exposure to beta-cypermethrin resulted in a decrease in the precursor material (trehalose, acetyl-CoA) and enzyme activities involved in sex pheromone biosynthesis, This, in turn, led to reduced pheromone production and female attractiveness, as well as diminished male detection abilities, ultimately resulted in a lowered likelihood of successful mating. This study enhances knowledge on how sublethal pesticide exposure affects pest biology and their mechanisms of action, providing a basis for including such impact in pest management strategies. [ABSTRACT FROM AUTHOR]

BACKGROUND: Extensive planting of transgenetic Bacillus thuringiensis crops has driven the evolution of pest resistance to Cry1Ac. Adjustment of cropping structures has promoted further outbreak of Helicoverpa armigera in China. To control this pest, a combination of pyramiding RNA interference (RNAi) and Cry2Ab is considered a promising strategy for countering cross-resistance and enhancing the toxicity of Cry2Ab to cotton bollworm. We explored the possibility of using calcineurin (CAN) as a target RNAi gene, because it is involved in cotton bollworm responses to the toxicity of Cry2Ab. RESULTS: Cry2Ab treatment led to a significant increase in HaCAN mRNA level and HaCAN activity. Suppression of HaCAN activity due to RNAi-mediated knockdown of HaCAN increased the susceptibility of midgut cells to Cry2Ab. The increase in HaCAN activity shown by heterologous expression of HaCAN reduced the cytotoxicity of Cry2Ab to Sf9 cells. Moreover, ingestion of HaCAN-specific inhibitor FK506 increased the toxicity of Cry2Ab in larvae. Interestingly, HaCAN does not function as a Cry2Ab direct binding protein that participates in Cry2Ab toxicity. CONCLUSIONS: The results in this study provide evidence that suppression of HaCAN not only affected the development of the cotton bollworm, but also enhanced the toxicity of Cry2Ab to the pest. HaCAN is therefore an important candidate gene in cotton bollworm that can be targeted for pest control when the pest infests RNAi+Cry2Ab crops. Meanwhile, the mechanism of action of HaCAN in Cry2Ab toxicity suggested that protein dephosphorylation was involved. [ABSTRACT FROM AUTHOR]

Trehalose is the major "blood sugar" of insects and it plays a crucial role in energy supply and as a stress protectant. The hydrolysis of trehalose occurs only under the enzymatic control of trehalase (Treh), which plays important roles in growth and development, energy supply, chitin biosynthesis, and abiotic stress responses. Previous reports have revealed that the vital hormone 20-hydroxyecdysone (20E) regulates Treh, but the detailed mechanism underlying 20E regulating Treh remains unclear. In this study, we investigated the function of HaTreh1 in Helicoverpa armigera larvae. The results showed that the transcript levels and enzymatic activity of HaTreh1 were elevated during molting and metamorphosis stages in the epidermis, midgut, and fat body, and that 20E upregulated the transcript levels of HaTrehl through the classical nuclear re-ceptor complex EcR-B1/USP1. HaTreh1 is a mitochondria protein. We also found that knockdown of HaTreh1 in the fifth- or sixth-instar larvae resulted in weight loss and increased mortality. Yeast two-hybrid, coimmunoprecipitation, and glutathione-S-transferase (GST) pull-down experiments demonstrated that HaTreh1 bound with ATP synthase subunit alpha (HaATPs-α) and that this binding increased under 20E treatment. In addition, 20E enhanced the transcript level of HaATPs-α and ATP content. Finally, the knockdown of HaTreh1 or HaATPs-α decreased the induction effect of 20E on ATP content. Altogether, these findings demonstrate that 20E controls ATP production by up-regulating the binding of HaTreh1 to HaATPs-α in H. armigera. [ABSTRACT FROM AUTHOR]