Identification and functional analysis of serine protease inhibitor gene family of Eocanthecona furcellata (Wolff).

The predatory natural enemy Eocanthecona furcellata plays a crucial role in agricultural ecosystems due to its effective pest control measures and defensive venom. Predator venom contains serine protease inhibitors (SPIs), which are the primary regulators of serine protease activity and play key roles in digestion, development, innate immunity, and other physiological regulatory processes. However, the regulation mechanism of SPIs in the salivary glands of predatory natural enemies is still unknown. In this study, we sequenced the transcriptome of E. furcellata salivary gland and identified 38 SPIs genes named EfSPI1∼EfSPI38. Through gene structure, multiple sequence alignment and phylogenetic tree analysis, real-time quantitative PCR (RT-PCR) expression profiles of different developmental stages and different tissues were analyzed. RNAi technology was used to explore the gene function of EFSPI20. The results showed that these 38 EfSPIs genes contained 8 SPI domains, which were serpin, TIL, Kunitz, Kazal, Antistasin, Pacifastin, WAP and A2M. The expression profile results showed that the expression of different types of EfSPIs genes was different at different developmental stages and different tissues. Most of the EfSPIs genes were highly expressed in the egg stage. The EfSPI20, EfSPI21, EfSPI22, and EfSPI24 genes of the Pacifastin subfamily and the EfSPI35 gene of the A2M subfamily were highly expressed in the nymphal and adult stages, which was consistent with the RT-qPCR verification results. These five genes are positively correlated with each other and have a synergistic effect on E. furcellata, and they were highly expressed in salivary glands. After interfering with the expression of the EfSPI20 gene, the survival rate and predatory amount of male and female adults were significantly decreased. Taken together, we speculated some EfSPIs may inhibit trypsin, chymotrypsin, and elastase, and some EfSPIs may be involved in autoimmune responses. EfSPI20 was essential for the predation and digestion of E. furcellata, and the functions of other EfSPIs were discussed. Our findings provide valuable insights into the diversity of EfSPIs in E. furcellata and the potential functions of regulating their predation, digestion and innate immunity, which may be of great significance for developing new pest control strategies.

Rapid cold hardening and cold acclimation promote cold tolerance of oriental fruit fly, Bactrocera dorsalis (Hendel) by physiological substances transformation and cryoprotectants accumulation.

Insect response to cold stress is often associated with adaptive strategies and chemical variation. However, low-temperature domestication to promote the cold tolerance potential of Bactrocera dorsalis and transformation of main internal substances are not clear. Here, we use a series of low-temperature exposure experiments, supercooling point (SCP) measurement, physiological substances and cryoprotectants detection to reveal that pre-cooling with milder low temperatures (5 and 10°C) for several hours (rapid cold hardening) and days (cold acclimation) can dramatically improve the survival rate of adults and pupae under an extremely low temperature (-6.5°C). Besides, the effect of rapid cold hardening for adults could be maintained even 4 h later with 25°C exposures, and SCP was significantly declined after cold acclimation. Furthermore, content of water, fat, protein, glycogen, sorbitol, glycerol and trehalose in bodies were measured. Results showed that water content was reduced and increased content of proteins, glycogen, glycerol and trehalose after two cold domestications. Our findings suggest that rapid cold hardening and cold acclimation could enhance cold tolerance of B. dorsalis by increasing proteins, glycerol, trehalose and decreasing water content. Conclusively, identifying a physiological variation will be useful for predicting the occurrence and migration trend of B. dorsalis populations.

Identification and expression analysis of heat shock protein family genes of gall fly (Procecidochares utilis) under temperature stress.

Heat shock proteins (HSP) are molecular chaperones involved in many normal cellular processes and environmental stresses. At the genome-wide level, there were no reports on the diversity and phylogeny of the heat shock protein family in Procecidochares utilis. In this study, 43 HSPs were identified from the genome of P. utilis, including 12 small heat shock proteins (sHSPs), 23 heat shock protein 40 (DNAJs), 6 heat shock protein 70 (HSP70s), and 2 heat shock protein 90 (HSP90s). The characteristics of these candidates HSP genes were analyzed by BLAST, and then phylogenetic analysis was carried out. Quantitative real-time PCR (qRT-PCR) was used to analyze the spatiotemporal expression patterns of sHSPs and HSP70s in P. utilis after temperature stress. Results showed that most sHSPs could be induced under heat stress during the adult stage of P. utilis, while a few HSP70s could be induced at the larval stage. This study provides an information framework for the HSP family of P. utilis. Moreover, it lays an important foundation for a better understanding of the role of HSP in the adaptability of P. utilis to various environments.

Azadirachtin inhibits the development and metabolism of the silk glands of Spodoptera frugiperda and affects spinning behavior.

BACKGROUND: Spodoptera frugiperda is a major agricultural pest, and the dispersal of its larvae by spinning silk is one of the causes of crop damage. At present, there are relatively few reports of pest control that affect larvae spinning silk. In this study, the effect of spinning behavior of the S. frugiperda larvae was investigated through a series of experiments. RESULTS: The 3rd instar larvae of S. frugiperda were exposed to azadirachtin, and the pathological changes in the silk glands of S. frugiperda and the differences in their metabolites were analyzed by scanning electron microscopy, histological sectioning, transmission electron microscopy and metabolomics. The results showed that azadirachtin could affect the silk gland of S. frugiperda. After 48 h of treatment with azadirachtin, the silk gland lumen of S. frugiperda appeared vacuolated. KEGG showed that 31 different metabolites were identified, of which 12 were upregulated and 19 were downregulated. These metabolites were enriched in 15 different metabolic pathways, which indicated that the silk gland of S. frugiperda was closely related to the formation of fatty acids and energy metabolism for the silk formation process. CONCLUSIONS: This study provides a preliminary report of the effect of azadirachtin on the spinning behavior of the S. frugiperda larvae. Metabolomic results indicated that histidine, glycine and leucine, which are related to serine protein synthesis, were down-regulated. Azadirachtin can damage the silk glands of S. frugiperda and thus affect spinning behavior. This provides the basis for the control of S. frugiperda by spinning silk. © 2022 Society of Chemical Industry.

Novel application of biodegradable chitosan in agriculture: Using green nanopesticides to control Solenopsis invicta.

Botanical pesticides are biological pesticides that are environment friendly. However, their instability and short persistence limit their application. In this study, pH sensitive chitosan based rotenone (Rot) nanoparticles (CS/CMCS/Rot-NPs) were prepared using chitosan and carboxymethyl chitosan to take advantage of the acidic nature of the red fire ant midgut. Chitosan based nanoparticles showed photoprotective and slow sustained release effects on Rot and significantly increased the insecticidal activity of Rot against red fire ants. The 24-96hLC50 of CS/CMCS/Rot-NPs against red fire ants was 3.28-6.84 fold that of Rot. The CS/CMCS/Rot-NPs significantly reduced the venom alkaloid content of red fire ants and their living environment and weakened their survival by increasing their survival cost in the ecological environment. Nanotechnology combined with botanical pesticides can be used as a novel, safe, effective, and ecofriendly method to control red fire ants.

Metabolic Changes in Larvae of Predator Chrysopa sinica Fed on Azadirachtin-Treated Plutella xylostella Larvae.

Biological control is a key component of integrated pest management (IPM). To suppress pests in a certain threshold, chemical control is used in combination with biological and other control methods. An essential premise for using pesticides in IPM is to ascertain their compatibility with beneficial insects. Chrysopa sinica (Neuroptera: Chrysopidae) is an important predator of various pests and used for pest management. This study was intended to analyze metabolic changes in C. sinica larvae after feeding on azadirachtin-treated Plutella xylostella (Lepidoptera, Plutellidae) larvae through a non-targeted LC-MS (Liquid chromatography-mass spectrometry) based metabolomics analysis. Results showed that C. sinica larvae did not die after consuming P. xylostella larvae treated with azadirachtin. However, their pupation and eclosion were adversely affected, resulting in an impairment in the completion of their life cycle. Feeding C. sinica larvae with azadirachtin-treated P. xylostella larvae affected over 10,000 metabolites across more than 20 pathways, including the metabolism of amino acids, carbohydrates, lipid, cofactors, and vitamins in C. sinica larvae, of which changes in amnio acid metabolism were particularly pronounced. A working model was proposed to illustrate differential changes in 20 metabolites related to some amino acid metabolisms. Among them, 15 were markedly reduced and only five were elevated. Our results suggest that azadirachtin application may not be exclusively compatible with the use of the predator C. sinica for control of P. xylostella. It is recommended that the compatibility should be evaluated not only based on the survival of the predatory insects but also by the metabolic changes and the resultant detrimental effects on their development.

Residue and dissipation of two formulations of emamectin benzoate in tender cowpea and old cowpea and a risk assessment of dietary intake.

The dissipation and residue levels of emamectin benzoate emulsifiable concentrate (EC) and microemulsion (ME) formulations in tender cowpeas and old cowpeas were investigated under field conditions. The decline curves of emamectin benzoate residues in cowpea corresponded to first-order kinetics. The dissipation rate of emamectin benzoate in tender cowpeas was faster than that in old cowpeas. The half-lives of the EC were 1.34-1.39 d and 1.74-2.31 d in tender cowpea and old cowpea, respectively. For the ME, the half-lives were 1.39-1.51 d and 2.08-2.67 d, respectively. The risk of adult intake of emamectin benzoate from cowpea is within the acceptable limits of the human body. Compared to tender cowpeas, the risk of eating old cowpeas is higher. Emamectin benzoate (EC) is recommended for cowpeas when the intention is to harvest tender cowpeas, while both formulations are acceptable for cowpeas when the intention is to harvest old cowpeas.

Insecticidal efficacy and mechanism of nanoparticles synthesized from chitosan and carboxymethyl chitosan against Solenopsis invicta (Hymenoptera: Formicidae).

The efficacy and mode of action of biodegradable chitosan (CS) and carboxymethyl chitosan (CMCS) organic polymer nanoparticles (NPs) on insects were studied. The prepared CS/CMCS-NPs were spherical with a particle size of 142.1 ± 2.0 nm. The swelling test showed that they were pH-sensitive, and the swelling rate was 554 % at pH 4.5. It was found that CS/CMCS-NPs had insecticidal efficacy against red fire ants (S. invicta). The mortality of red fire ants on the 6th day after treatment with 0.2 % and 0.06 % CS/CMCS-NPs suspensions was 98.33 ± 1.67 % and 48.33 ± 3.33 %, respectively. After CS/CMCS-NPs treatment, the food intake, growth, and development of red fire ants were inhibited; the midgut was significantly expanded; and the activity of digestive enzymes in the midgut was decreased. Our findings suggest that CS/CMCS-NPs mainly inhibited the digestion function of the midgut, leading to the death of red fire ants.

Azadirachtin directly or indirectly affects the abundance of intestinal flora of Spodoptera litura and the energy conversion of intestinal contents mediates the energy balance of intestine-brain axis, and along with decreased expression CREB in the brain neurons.

Azadirachtin is a good growth inhibitor for Lepidopteran larvae, but its effect on the brain neurons, intestinal flora and intestinal contents caused by the growth inhibition mechanism has not been reported yet. This study explored the mechanism of azadirachtin on the growth and development of Spodoptera litura larvae and brain neurons through three aspects: intestinal pathology observation, intestinal flora sequencing, and intestinal content analysis. The results showed that the treatment of azadirachtin led to the pathological changes in the structure of the midgut and the goblet cells in the intestinal wall cells to undergo apoptosis. Changes in the host environment of the intestinal flora lead to changes in the abundance value of the intestinal flora, showing an increase in the abundance value of harmful bacteria such as Sphingomonas and Enterococcus, as well as an increase in the abundance value of excellent flora such as Lactobacillus and Bifidobacterium. Changes in the abundance of intestinal flora will result in changes in intestinal contents and metabolites. The test results show that after azadirachtin treatment, the alkane compounds in the intestinal contents of the larvae are greatly reduced, and the number of the long carbon chain and multi-branched hydrocarbon compounds is increased, unsaturated fatty acids, silicon­oxygen compounds and ethers. The production of similar substances indicates that azadirachtin has an inhibitory effect on digestive enzymes in the intestines, which results in the inhibition of substance absorption and energy transmission, and ultimately the inhibition of larval growth and brain neurons.

Treating green pea aphids, Myzus persicae, with azadirachtin affects the predatory ability and protective enzyme activity of harlequin ladybirds, Harmonia axyridis.

As a natural enemy of green peach aphids, harlequin ladybirds, Harmonia axyridis Pallas (Coleoptera: Coccinellidae), are also indirectly affected by azadirachtin. In this study, we evaluated the effects of ladybird exposure to azadirachtin through azadirachtin-treated aphids. About 2 mg/L azadirachtin treated aphid can deliver the azadirachtin to ladybird larvae in 12 and 24 h. And azadirachtin treatment affected the rate at which fourth instar larvae and adult ladybirds preyed on aphids. Furthermore, the antifeedant effect increased with increasing azadirachtin concentrations. Twelve hours after exposing fourth instar ladybird larvae to aphids treated with 10 mg/L azadirachtin, the antifeedant effect was 47.70%. Twelve hours after exposing adult ladybirds to aphids treated with 2 mg/L azadirachtin, the antifeedant effect was 67.49%. Forty-eight hours after exposing ladybird larvae to azadirachtin-treated aphids, their bodyweights were 8.37 ± 0.044 mg (2 mg/L azadirachtin), 3.70 ± 0.491 mg (10 mg/L azadirachtin), and 2.39 ± 0.129 mg (50 mg/L azadirachtin). Treatment with azadirachtin affected the ability of ladybirds to prey on aphids. The results indicated that the instant attack rate of ladybird larvae and adults and the daily maximum predation rate were reduced by azadirachtin treatment. Superoxide dismutase (SOD), peroxidase (POD), and peroxide (CAT) enzyme activities of ladybirds were affected after feeding on aphids treated with azadirachtin. Azadirachtin has certain antifeedant effects on ladybirds and affects the ability of ladybirds to prey on aphids and the activities of SOD, POD, and CAT enzymes, which results in inhibition of normal body development.

Azadirachtin downregulates the expression of the CREB gene and protein in the brain and directly or indirectly affects the cognitive behavior of the Spodoptera litura fourth-instar larvae.

BACKGROUND: Azadirachtin has the potential to be used for pest control. Nevertheless, few studies have investigated the effects of azadirachtin on the cognitive behavior of pests. In this study, expression of the cAMP response element-binding protein (CREB) and its gene were studied via a series of experiments in Spodoptera litura larvae treated with azadirachtin. RESULTS: RNA-Seq analysis of S. litura larvae treated with azadirachtin was undertaken. According to Kyoto Encyclopedia of Genes and Genomes analysis, the top 20 enriched pathways included neuroactive ligand-receptor interaction pathways, with seven significantly differentially expressed genes including CREB. Quantitative real time polymerase chain reaction (qRT-PCR) results indicated that the CREB gene was expressed during all developmental stages of S. litura, but relative expression of the CREB gene was significantly downregulated after treatment with azadirachtin. Grayscale statistical analysis also showed that expression levels of protein kinase A (PKA), extracellular signal-regulated kinase (ERK) and CREB proteins were significantly downregulated after treatment with azadirachtin. Moreover, RNA interference results showed that the effect of azadirachtin on the cognitive behavior of S. litura was consistent with that seen after interfering with CREB. In addition, larval selectivity to addictive odor sources was reduced, and the initial reaction time was increased. CONCLUSIONS: This study clarified that azadirachtin can affect the cognitive behavior of S. litura and treatment with azadirachtin resulted in a downregulation of gene and protein expression of CREB and its pathway proteins. © 2020 Society of Chemical Industry.

Toxicity and Sublethal Effects of Autumn Crocus (Colchicum autumnale) Bulb Powder on Red Imported Fire Ants (Solenopsis invicta).

Autumn crocus (Colchicum autumnale L.) is a medicinal plant as it contains high concentrations of colchicine. In this study, we reported that the ground powder of autumn crocus bulb is highly toxic to invasive Solenopsis invicta Buren, commonly referred to as red imported fire ants (RIFAs). Ants fed with sugar water containing 5000 mg/L of bulb powder showed 54.67% mortality in three days compared to 45.33% mortality when fed with sugar water containing 50 mg/L of colchicine. Additionally, the effects of short-term feeding with sugar water containing 1 mg/L of colchicine and 100 mg/L of autumn crocus bulb powder were evaluated for RIFAs' colony weight, food consumption, and aggressiveness, i.e., aggregation, grasping ability, and walking speed. After 15 days of feeding, the cumulative colony weight loss reached 44.63% and 58.73% due to the sublethal concentrations of colchicine and autumn crocus bulb powder, respectively. The consumption of sugar water and mealworm (Tenebrio molitor L.) was substantially reduced. The aggregation rates decreased 48.67% and 34.67%, grasping rates were reduced to 38.67% and 16.67%, and walking speed decreased 1.13 cm/s and 0.67 cm/s as a result of the feeding of the two sublethal concentrations of colchicine and autumn crocus bulb powder, respectively. Our results for the first time show that powder derived from autumn crocus bulbs could potentially be a botanical pesticide for controlling RIFAs, and application of such a product could be ecologically benign due to its rapid biodegradation in the environment.

Plants in the Genus Tephrosia: Valuable Resources for Botanical Insecticides.

Synthetic insecticides are effective in controlling insect pests but can also harm nontarget organisms and the environment. During the last 40 years, there has been an increasing interest in alternative insecticides, particularly those derived from plants, commonly known as botanical insecticides. However, commercially available botanical insecticides remain limited. Rotenone is one of the earliest identified compounds and was used as fish poison and pest management. Due to its link with Parkinson disease, the use of rotenone was banned in many developed countries. Rotenone used to be isolated from Derris spp. and Lonchocarpus spp., and it can also be isolated from Tephrosia species. In this article, we present basic botanical information on selected Tephrosia species and their major compounds related to insecticidal activities and highlight the current use of extracts derived from some species, Tephrosia vogelii in particular, for control of insect pests in stored grains and crop production. The crude extracts contain multiple bioactive compounds, mainly rotenone, deguelin, rotenolone, and tephrosin, which act in either additive or synergistic fashion, resulting in effective control of insect pests. There are about 400 species in the genus Tephrosia, and species and even strains or variants vary greatly in these active compounds. We argue that a systematic evaluation of bioactive compounds in different species are needed, and species or strains with high insecticidal activities should be selected for use in the sustainable control of insect pests.

Different lethal treatments induce changes in piperidine (1,1'-(1,2-ethanediyl)bis-) in the epidermal compounds of red imported fire ants and affect corpse-removal behavior.

Corpse-removal behavior of the red imported fire ant (RIFA) and the effects of lethal substances on RIFA signal communication were investigated in this study. The RIFA corpses, obtained through freezing, ether, 0.25 mg/L thiamethoxam, and starvation to death treatments, and naturally dead red fire ants were subjected to gas chromatography-mass spectrometry to identify the cuticular hydrocarbon profiles that had an effect on the corpse-removal behavior. The results showed that lethal toxic substances altered the epidermal compounds of RIFA and affected their corpse-removal behavior. Lethal toxic substances increased the number of worker touches with corpses and identification time of corpses. In addition, the content of piperidine (1,1'-(1,2-ethanediyl)bis-) on the surface of the corpse was different following the various treatments. Contamination with toxic substances resulted in the increased secretion of piperidine and led to increased identification time of corpses, number of touch with corpses, and total time for removal of corpses. Piperidine content was higher under conditions of natural death (4.67 ± 0.55%) and with thiamethoxam (10.43 ± 0.78%), freezing (0.83 ± 0.25%), and ether treatment (12.50 ± 0.70%) than under starvation treatment (0). The higher content of piperidine led to a longer number of touches with corpses and identification time. Piperidine compounds may be an element in warning information, which could affect the occurrence of different corpse-removal behaviors.

Dissipation and distribution of difenoconazole in bananas and a risk assessment of dietary intake.

The dissipation and terminal residues of difenoconazole in whole bananas and pulp were investigated under field conditions. The residual levels of difenoconazole in various parts of bananas grown in Guangdong, Hainan and Yunnan were determined by a GC-ECD detection method after simple, rapid pretreatment. The mean recovery was 80.66~107.40%, and the relative standard deviation was 3.36~9.84%. The results showed that the half-lives of difenoconazole in whole bananas and in the pulp were 12.16~13.33 days and 17.77~20.38 days, respectively. At harvest intervals of 28 and 35 days after the last application, the terminal residues of difenoconazole in whole bananas and pulp were 0.45~0.84 mg/kg and 0.19~0.37 mg/kg, respectively, which were lower than the maximum residue level established in China. The distribution of difenoconazole in banana pulp and peels was studied. The results showed that until harvesting, the residue in the peels was always 2.19~12.30 times larger than that in the pulp. Difenoconazole was mainly absorbed by the banana peels but did not easily penetrate into the pulp. Based on dietary risk assessment results, the residual levels of difenoconazole at the sampling interval of 28 days after the last application were within acceptable limits for chronic and acute dietary risks in different populations in China. This study can provide a reference for the safe and rational use of difenoconazole as a fungicide and for the future research and application of banana pulp and peels.

The comparative metabolic response of Bactrocera dorsalis larvae to azadirachtin, pyriproxyfen and tebufenozide.

Azadirachtin, as the most promising and effective botanical insecticide, exhibits significant growth inhibition activity against agricultural and forestry pests. However, its biochemical effects at the metabolic level compared with those of other insect growth regulators have not been studied. Therefore, in this study, a GC-MS based untargeted metabolomics approach was applied to compare azadirachtin with pyriproxyfen (a juvenile hormone analog) and tebufenozide (a molting hormone analog) in terms of their metabolic effects on Bactrocera dorsalis larvae. The bioactivity of azadirachtin against B. dorsalis larvae was significantly different than those of pyriproxyfen and tebufenozide. A total of 693 mass features were recognized, and 112 metabolites were identified in this study. The results showed that a total of 16, 13 and 10 differentially regulated metabolites corresponding to 12, 5 and 8 pathways occur in Aza versus CK, Pyr versus CK and Teb versus CK group, respectively. Further analysis showed that 6 differentially regulated metabolites corresponding to 5 key pathways could be the primary differential metabolic response of B. dorsalis larvae to the three insect growth regulators. The pathways were myo-inositol corresponding to ascorbate and aldarate metabolism as the specific response of B. dorsalis larvae to azadirachtin; xylitol, xylulose and 3-aminopropionitrile corresponding to pentose and glucuronate interconversions, and cyanoamino acid metabolism as the common responses to azadirachtin and pyriproxyfen; and 3-hydroxypropionic acid and beta-alanine corresponding to propanoate metabolism and beta-alanine metabolism as the specific responses to tebufenozide. The results showed that the metabolic response of B. dorsalis larvae to azadirachitin is closer to that of pyriproxyfen than tebufenozide. The differentially regulated metabolites and pathways responsible for this difference are discussed.

Antifeeding effects of azadirachtin on the fifth instar Spodoptera litura larvae and the analysis of azadirachtin on target sensilla around mouthparts.

To clarify the types, number, and distribution of sensilla on the head of the fifth instar Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) larvae and identify the main sensilla of azadirachtin acting on larvae, scanning electron microscopy was used to study the morphology of the head and sensilla on the mouthparts. The four sensilla-sensillum basiconicum, sensillum chaeticum, sensillum styloconicum, and sensillum trichodeum-on the head of the fifth instar larvae were treated with 0, 0.1, 0.5, 1, 2, and 4 mg/kg azadirachtin by a microdrop method. The larvae showed an obvious antifeeding effect with azadirachtin. And higher the concentration of azadirachtin, the more obvious the phenomenon of antifeeding activity. The sensillum styloconicum and the sensillum trichodeum were the main sensilla for azadirachtin. When 1 mg/kg azadirachtin was used to treat sensillum styloconicum and sensillum basiconicum, the fifth instar larvae of S. litura showed obvious antifeedant activity and the cumulative feed intake for 24 hr was no more than 30% of the leaf area. Quantitative reverse-transcription polymerase chain reaction verified the expression patterns of some Grs, indicating that Grst43a was upregulated by 1.3- and 3.9-fold, Gor24 was upregulated by 2.5- and 3.3-fold, Gr5a was downregulated by 0.6-fold and upregulated by 2.0-fold, and Gr28a was downregulated by 0.8-fold and upregulated by 3.6-fold upon treatment with 0.5 mg/kg and 1 mg/kg azadirachtin in 24 hr. Gr genes participated in the identification of bitterness and we speculated that Gr genes may indirectly lead to the occurrence of antifeeding behavior.