Advances in Aptamer-Based Conjugate Recognition Techniques for the Detection of Small Molecules in Food.

Small molecules are significant risk factors for causing food safety issues, posing serious threats to human health. Sensitive screening for hazards is beneficial for enhancing public security. However, traditional detection methods are unable to meet the requirements for the field screening of small molecules. Therefore, it is necessary to develop applicable methods with high levels of sensitivity and specificity to identify the small molecules. Aptamers are short-chain nucleic acids that can specifically bind to small molecules. By utilizing aptamers to enhance the performance of recognition technology, it is possible to achieve high selectivity and sensitivity levels when detecting small molecules. There have been several varieties of aptamer target recognition techniques developed to improve the ability to detect small molecules in recent years. This review focuses on the principles of detection platforms, classifies the conjugating methods between small molecules and aptamers, summarizes advancements in aptamer-based conjugate recognition techniques for the detection of small molecules in food, and seeks to provide emerging powerful tools in the field of point-of-care diagnostics.

RT-RPA-PfAgo System: A Rapid, Sensitive, and Specific Multiplex Detection Method for Rice-Infecting Viruses.

The advancement in CRISPR-Cas biosensors has transmuted the detection of plant viruses owing to their rapid and higher sensitivity. However, false positives and restricted multiplexing capabilities are still the challenges faced by this technology, demanding the exploration of novel methodologies. In this study, a novel detection system was developed by integrating reverse transcriptome (RT) techniques with recombinase polymerase isothermal amplification (RPA) and Pyrococcus furiosus Argonaute (PfAgo). The RT-RPA-PfAgo system enabled the simultaneous detection of rice ragged stunt virus (RRSV), rice grassy stunt virus (RGSV), and rice black streaked dwarf virus (RBSDV). Identifying targets via guide DNA without being hindered by protospacer adjacent motif sequences is the inherent merit of PfAgo, with the additional advantage of it being simple, cost-effective, and exceptionally sensitive, with detection limits between 3.13 and 5.13 copies/µL, in addition to it effectively differentiating between the three distinct viruses. The field evaluations were also in accordance with RT-PCR methods. The RT-RPA-PfAgo system proved to be a robust, versatile, highly specific, and sensitive method with great potential for practicality in future plant virus diagnostics.

Establishment of a Rapid Detection Method for Yeast-like Symbionts in Brown Planthopper Based on Droplet Digital PCR Technology.

The brown planthopper Nilaparvata lugens (Stål) (BPH) is a typical monophagous sucking rice pest. Over the course of their evolution, BPH and its symbionts have established an interdependent and mutually beneficial relationship, with the symbionts being important to the growth, development, reproduction, and variation in virulence of BPH. Yeast-like symbionts (YLS), harbored in the abdomen fat body cells of BPH, are vital to the growth and reproduction of the host. In recent research, the symbionts in BPH have mainly been detected using blood cell counting, PCR, real-time quantitative PCR, and other methods. These methods are vulnerable to external interference, cumbersome, time consuming and laborious. Droplet digital PCR (ddPCR) does not need a standard curve, can achieve absolute quantification, does not rely on Cq values, and is more useful for analyzing copy number variation, gene mutations, and relative gene expression. A rapid detection method for the YLS of BPH based on ddPCR was established and optimized in this study. The results showed that the method's limits of detection for the two species of YLS (Ascomycetes symbionts and Pichia guilliermondii) were 1.3 copies/µL and 1.2 copies/µL, respectively. The coefficient of variation of the sample repetition was less than 5%; therefore, the ddPCR method established in this study had good sensitivity, specificity, and repeatability. It can be used to detect the YLS of BPH rapidly and accurately.

A Novel Cuticular Protein-like Cpr21L Is Essential for Nymph Survival and Male Fecundity in the Brown Planthopper.

Cuticular proteins (CPs) are a large family and perform a variety of functions. However, the physiological roles of cuticle protein 21-like (Cpr21L) in the brown planthopper (Nilaparvata lugens, BPH), one of the most destructive insect pests of rice, are largely unclear. In this study, Cpr21L was revealed to be expressed in both BPH nymphs and adults, and the mRNA expression level was much higher in male adults than female adults. Spatially, the expression of Cpr21L in the testis was higher than in the ovary. The RNA interference (RNAi) of Cpr21L seriously decreased nymph survival, and no individual survived 8 days post-dsCpr21L injection. The RNAi of Cpr21L in adults also decreased the fertility of males, especially in the dsCpr21L♂ × dsGFP♀ group. The average number of eggs laid by one female in this group significantly decreased by 50.1%, and the eggs' hatchability decreased from 76.5% to 23.8% compared with the control (dsGFP♂ × dsGFP♀). Furthermore, observations under a stereomicroscope showed that the RNAi of Cpr21L severely impaired the development of the testes. Therefore, Cpr21L is essential for the nymphal survival and male fecundity of BPH, thus providing a possible target for pest control.

Effects of Secondary Metabolites of Rice on Brown Planthopper and Its Symbionts.

The brown planthopper Nilaparvata lugens (Stål) (BPH) is a main rice pest in China and many other Asian countries. In the control of BPH, the application of insect-resistant rice has proven to be quite effective. Secondary metabolites are essential weapons in plants' defense against phytophagous insects. Studies have found that differences in the content of secondary metabolites play a crucial role in determining whether rice exhibits resistance or susceptibility to BPH. Simultaneously, symbionts are essential to the BPH. Nevertheless, there is limited research on the impact of secondary metabolites on the symbionts within BPH. Therefore, investigating the influence of secondary metabolites on both BPH and their symbionts is significant for the control of BPH. In this experiment, newly emerged female adults of BPH were fed artificial diets containing 10 different secondary metabolites. The results indicated that methyl jasmonate had inhibitory effects on the survival rate, weight gain, and reproductive capacity of BPH. Using qPCR methods, it was discovered that the number of symbiotic fungi (Ascomycetes symbionts) within BPH significantly decreased under methyl jasmonate stress. In conclusion, this experiment has preliminarily revealed the inhibitory effects of methyl jasmonate on BPH and its symbionts, demonstrating its potential for controlling BPH.

Rice Defense against Brown Planthopper Partially by Suppressing the Expression of Transferrin Family Genes of Brown Planthopper.

Transferrins are multifunctional proteins, but their role in the interaction of rice and brown planthopper (BPH) remains unclear. In this study, the full-length cDNA of transferrin genes NlTsf1, NlTsf2, and NlTsf3 was cloned. Reverse transcription quantitative polymerase chain reaction showed that the expressions of NlTsf1 and NlTsf3 were significantly suppressed in BPH reared on the resistant rice R1 by 68.0 and 86.7%, respectively, compared with that on the susceptible S9. The survival rate decreased to 3.3% for dsNlTsf3-treated nymphs, to 58.9% for dsNlTsf1, and to 56.7% for dsNlTsf2 on day 11. RNAi of NlTsf3 against females largely reduced the number of eggs by 99.4%, and it decreased by 48.6% for dsNlTsf1 but did not significantly decrease for dsNlTsf2. Collectively, NlTsf1, NlTsf2, and NlTsf3 are essential for the survival and fecundity of BPH and are differentially involved in the interaction between rice and BPH. Therefore, NlTsf1 and NlTsf3 may be used as targets to control BPH.

Detection of Yeast-like Symbionts in Brown Planthopper Reared on Different Resistant Rice Varieties Combining DGGE and Absolute Quantitative Real-Time PCR.

The brown planthopper (BPH), Nilaparvata lugens, is a serious pest of rice throughout Asia. Yeast-like symbionts (YLS) are endosymbionts closely linked with the development of BPH and the adapted mechanism of BPH virulence to resistant plants. In this study, we used semi-quantitative DGGE and absolute quantitative real-time PCR (qPCR) to quantify the number of the three YLS strains (Ascomycetes symbionts, Pichia-like symbionts, and Candida-like symbionts) that typically infect BPH in the nymphal stages and in newly emerged female adults. The quantities of each of the three YLS assessed increased in tandem with the developing nymphal instar stages, peaking at the fourth instar stage, and then declined significantly at the fifth instar stage. However, the amount of YLS present recovered sharply within the emerging adult females. Additionally, we estimated the quantities of YLS for up to eight generations after their inoculation onto resistant cultivars (Mudgo, ASD7, and RH) to reassociate the dynamics of YLS with the fitness of BPH. The minimum number of each YLS was detected in the second generation and gradually increased from the third generation with regard to resistant rice varieties. In addition, the Ascomycetes symbionts of YLS were found to be the most abundant of the three YLS strains tested for all of the development stages of BPH.

RNAi-mediated silencing of the autophagy-related gene NlATG3 inhibits survival and fecundity of the brown planthopper, Nilaparvata lugens.

BACKGROUND: The brown planthopper (BPH), Nilaparvata lugens, is a serious insect pest of rice. Autophagy and its related gene ATG3 play multiple roles in insects. However, information about the functions of ATG3 in BPH (NlATG3) is unavailable, and its potential as a target for pest control remains unclear. RESULTS: RT-qPCR results showed a relatively low expression of NlATG3 in 1st-4th-instar nymphs, which increased through 9-day-old adults. The expression of NlATG3 increased continuously in 1-day-old through 5-day-old eggs, whereas it decreased thereafter. The mRNA level of NlATG3 was markedly higher in the ovary (1.16) and head (1.00) compared to the rest body parts of BPH adults. Injecting nymphs with dsNlATG3 at doses from 62.5 to 250 ng per insect had strong lethal effect upon them. For the 5th-instar nymphs, all individuals died within 5 days after receiving the dsNlATG3, and importantly, no individual successfully molted. Transmission electron microscopy revealed the new cuticle of nymphs injected with dsNlATG3 became loose and curved, which is clearly different from that of the control. Correspondingly, the obvious vesicles in epidermal cells disappeared after dsNlATG3-treatment. RNAi of NlATG3 significantly reduced the total number of eggs laid per female as well as the eggs' hatchability, especially in the dsNlATG3♀ × dsGFP♂ group, whose total number of eggs laid per female largely decreased by 80.4%, and whose eggs' hatchability was significantly reduced from 95.7% to zero, when compared with the control (dsGFP♀ × dsGFP♂). CONCLUSION: NlATG3 is a promising target for developing RNAi-based insect management strategies. © 2021 Society of Chemical Industry.

Establishment of an Indirect Competitive Enzyme-Linked Immunosorbent Method for the Detection of Heavy Metal Cadmium in Food Packaging Materials.

Heavy metals in food packaging materials have been indicated to release into the environment at slow rates. Heavy metal contamination, especially that of cadmium (Cd), is widely acknowledged as a global environment threat that leads to continuous growing pollution levels in the environment. Traditionally, the detection of the concentration of Cd relies on expensive precision instruments, such as inductively coupled plasma mass spectrometry (ICP-MS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES). In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a specific monoclonal antibody was proposed to rapidly detect Cd. The half-inhibitory concentration and detection sensitivity of the anti-cadmium monoclonal antibody of the ic-ELISA were 5.53 ng mL-1 and 0.35 ng mL-1, respectively. The anti-Cd monoclonal antibody possessed high specificity while diagnosising other heavy metal ions, including Al (III), Ca (II), Cu (II), Fe (III), Hg (II), Mg (II), Mn (II), Pb (II), Zn (II), Cr (III) and Ni (II). The average recovery rates of Cd ranged from 89.03-95.81% in the spiked samples of packing materials, with intra- and inter-board variation coefficients of 7.20% and 6.74%, respectively. The ic-ELISA for Cd detection was applied on 72 food packaging samples that consisted of three material categories-ceramic, glass and paper. Comparison of the detection results with ICP-AES verified the accuracy of the ic-ELISA. The correlation coefficient between the ic-ELISA and the ICP-AES methods was 0.9634, demonstrating that the proposed ic-ELISA approach could be a useful and effective tool for the rapid detection of Cd in food packaging materials.

Fungicides Reduce the Abundance of Yeast-like Symbionts and Survival of White-Backed Planthopper Sogatella furcifera (Homoptera: Delphacidae).

The white-backed planthopper (WBPH) Sogatella furcifera is one of the most harmful pests of rice in Southeast Asia. The fat body of WBPH harbors intracellular yeast-like symbionts (YLS). YLS are vertically transmitted to WBPH offspring by transovarial infection. YLS play an important role in the WBPH life cycle. YLS diversity and function have been extensively studied in the brown planthopper (BPH) and small brown planthopper but not in WBPH, even though a novel strategy for controlling the BPH based on suppressing YLS has been proposed. Here, using denaturing gradient gel electrophoresis, we identified 12 unique fungal sequences among YLS of WBPH, and five of them represented uncultured fungi. We then fed WBPH with rice plants treated with different fungicides [70% propineb wettable powder (WP) (PR), 70% propamocarb hydrochloride aqueous solution (AS) (PH), 25% trifloxystrobin and 50% tebuconazole water-dispersible granules (WG) (TT), 40% pyrimethanil suspension concentrate (SC) (PY), and 50% iprodione SC (IP)] and evaluated their effects on YLS abundance and WBPH survival rate. Both YLS abundance and adult WBPH survival rate were significantly decreased upon feeding fungicide-treated rice plants, and exposure to 50% IP resulted in the strongest reduction. The abundance of two Sf-YLS species (Ascomycetes symbiotes and Cla-like symbiotes) was significantly reduced upon exposure to 50% IP. The counts of Ascomycetes symbiotes, the most abundant YLS species, were also suppressed by the other fungicides tested. In conclusion, 50% IP was the most effective fungicide, reducing YLS abundance and WBPH survival rate under controlled conditions, suggesting its potential use to control WBPH.

NlATG1 Gene Participates in Regulating Autophagy and Fission of Mitochondria in the Brown Planthopper, Nilaparvata lugens.

Autophagy plays multiple roles in regulating various physiological processes in cells. However, we currently lack a systematic analysis of autophagy and the autophagy-related gene 1 ATG1 in the brown planthopper (BPH, Nilaparvata lugens), one of the most destructive of the insect pests of rice. In this study, the full-length cDNA of an autophagy-related gene, NlATG1, was cloned from BPH. Real-time qPCR (RT-qPCR) revealed that this NlATG1 gene was expressed differently across developmental stages, at higher levels in nymphs but lower levels in adults. RNA interference with dsNlATG1 significantly decreased the mRNA level of the target gene to 14.6% at day 4 compared with that of the dsGFP control group. The survival of the dsNlATG1-treated group decreased significantly from day 4 onward, dropping to 48.3% on day 8. Examination using transmission electron microscopy (TEM) showed that epithelial cells of the BPH's midgut in the dsNlATG1-treated group had less autophagic vacuoles than did the dsGFP control, and knockdown of NlATG1 clearly inhibited the starvation-induced autophagy response in this insect. RNA interference of NlATG1 upregulated the NlFis1 gene involved in mitochondrial fission, leading to reductions in mitochondrial width and area. Furthermore, knockdown of NlATG1 also decreased the ATP content and accumulation of glycogen. Together, these results demonstrate that the NlATG1 gene participates in regulating autophagy and fission of mitochondria in the brown planthopper, making it a potentially promising target for pest control given its key role in autophagy, including maintaining the normal structure and function of mitochondria.

Schaftoside Interacts With NlCDK1 Protein: A Mechanism of Rice Resistance to Brown Planthopper, Nilaparvata lugens.

Brown planthopper (BPH) Nilaparvata lugens Stål is a serious insect pest of rice in Asian countries. Active compounds have close relationship with rice resistance against BPH. In this study, HPLC, MS/MS, and NMR techniques were used to identify active compounds in total flavonoids of rice. As a result, a BPH resistance-associated compound, Peak 1 in HPLC chromatogram of rice flavonoids, was isolated and identified as schaftoside. Feeding experiment with artificial diet indicated that schaftoside played its role in a dose dependent manner, under the concentration of 0.10 and 0.15 mg mL-1, schaftoside showed a significant inhibitory effect on BPH survival (p < 0.05), in comparison with the control. The fluorescent spectra showed that schaftoside has a strong ability to bind with NlCDK1, a CDK1 kinase of BPH. The apparent association constant KA for NlCDK1 binding with schaftoside is 6.436 × 103 L/mol. Docking model suggested that binding of schaftoside might affect the activation of NlCDK1 as a protein kinase, mainly through interacting with amino acid residues Glu12, Thr14 and Val17 in the ATP binding element GXGXXGXV (Gly11 to Val18). Western blot using anti-phospho-CDK1 (pThr14) antibody confirmed that schaftoside treatment suppressed the phosphorylation on Thr-14 site of NlCDK1, thus inhibited its activation as a kinase. Therefore, this study revealed the schaftoside-NlCDK1 interaction mode, and unraveled a novel mechanism of rice resistance against BPH.

Expression and RNA Interference of Ribosomal Protein L5 Gene in Nilaparvata lugens (Hemiptera: Delphacidae).

The ribosomal proteins play important roles in the growth and development of organisms. This study aimed to explore the function of NlRPL5 (GenBank KX379234), a ribosomal protein L5 gene, in the brown planthopper Nilaparvata lugens. The open reading frame of NlRPL5 was cloned from N. lugens based on a previous transcriptome analysis. The results revealed that the open reading frame of NlRPL5 is of 900 bp, encoding 299 amino acid residues. The reverse transcription quantitative PCR results suggested that the expression of NlRPL5 gene was stronger in gravid females, but was relatively low in nymphs, males, and newly emerged females. The expression level of NlRPL5 in the ovary was about twofolds of that in the head, thorax, or fat body. RNAi of dsNlRPL5 resulted in a significant reduction of mRNA levels, ∼50% decrease in comparison with the dsGFP control at day 6. Treatment of dsNlRPL5 significantly restricted the ovarian development, and decreased the number of eggs laid on the rice (Oryza sativa) plants. This study provided a new clue for further study on the function and regulation mechanism of NlRPL5 in N. lugens.

Roles of NlAKTIP in the Growth and Eclosion of the Rice Brown Planthopper, Nilaparvata lugens Stål, as Revealed by RNA Interference.

AKT-interacting protein (AKTIP) interacts with serine/threonine protein kinase B (PKB)/AKT. AKTIP modulates AKT's activity by enhancing the phosphorylation of the regulatory site and plays a crucial role in multiple biological processes. In this study, the full length cDNA of NlAKTIP, a novel AKTIP gene in the brown planthopper (BPH) Nilaparvata lugens, was cloned. The reverse transcription quantitive PCR (RT-qPCR) results showed that the NlAKTIP gene was strongly expressed in gravid female adults, but was relatively weakly expressed in nymphs and male adult BPH. In female BPH, treatment with dsAKTIP resulted in the efficient silencing of NlAKTIP, leading to a significant reduction of mRNA levels, about 50% of those of the untreated control group at day 7 of the study. BPH fed with dsAKTIP had reduced growth with lower body weights and smaller sizes, and the body weight of BPH treated with dsAKTIP at day 7 decreased to about 30% of that of the untreated control. Treatment of dsAKTIP significantly delayed the eclosion for over 7 days relative to the control group and restricted ovarian development to Grade I (transparent stage), whereas the controls developed to Grade IV (matured stage). These results indicated that NlAKTIP is crucial to the growth and development of female BPH. This study provided a valuable clue of a potential target NlAKTIP for inhibiting the BPH, and also provided a new point of view on the interaction between BPH and resistant rice.

Antifeedant and insecticidal effects of mandelic acid on the brown planthopper Nilaparvata lugens Stål.

To study the effects of mandelic acid (MA) on the brown planthopper (BPH), Nilaparvata lugens, the survival rate and behaviour of BPH fed on an artificial diet with different dosages of MA was observed. The survival rate of BPH decreased with the increase of the MA concentration and feeding time. In contrast to the control, the survival rate of BPH 72 h after feeding decreased significantly. Electrical penetration graph (EPG) data indicated that MA absorbed by the rice plant from Kimura B solution significantly affected the feeding behaviour of BPH. At the concentrations of 0.1, 0.5, and 1.0 mg/ml, duration of the phloem ingestion of BPH decreased from 115.34 min (control) to 30.41, 7.63, and 0.36 min, respectively. Periods of xylem ingestion of MA-treated BPH were significantly shorter than those of the control (50.44 min). Moreover, BPH spent more time walking around or being at rest on MA-treated rice plants, as well as in stylet activities. The GST (glutathione S-transferase) activity of BPH increased with the increasing MA concentration, while the GPX (glutathione peroxidases) activity did not change significantly. The results indicate that MA has an antifeedant and insecticidal effect on BPH.

Identification of two species of yeast-like symbiotes in the brown planthopper, Nilaparvata lugens.

To determine the species of the yeast-like symbionts (YLS) in the brown planthoppers (BPH), Nilaparvata lugens, YLS were first isolated and purified by ultracentrifugation from the fat bodies of BPH, and then 18S rDNA and internal transcribed spacer (ITS)-5.8S rDNA sequences of YLS were amplified with the different general primers for fungi. The results showed that the two different 18S and ITS-5.8S rDNA sequences of YLS were obtained. One 2291-bp DNA sequence, which contained 18S and ITS-5.8S rDNA, showed the high similarity to Cryptococcus and was named Cryp-Like symbiotes. Another 1248-bp DNA sequence, which contained a part of 18S and ITS-5.8S rDNA, showed the high similarity to Pichia guilliermondii and was named Pichia-Like symbiotes. It was further proved that Cryp- and Pichia-Like symbiotes existed in BPH through nested PCR with specific primers for two symbiotes and in situ hybridization analysis using digoxigenin-labeled probes. Our results showed that BPH harbored more than one species of eukaryotic YLS, which suggested that diversity of fungal endosymbiotes may be occurred in planthoppers, just like bacterial endosymbiotes.

Herbivore-induced callose deposition on the sieve plates of rice: an important mechanism for host resistance.

The brown planthopper (Nilaparvata lugens Stål; BPH) is a specialist herbivore on rice (Oryza sativa) that ingests phloem sap from the plant through its stylet mouthparts. Electronic penetration graphs revealed that BPH insects spent more time wandering over plants carrying the resistance genes Bph14 and Bph15, but less time ingesting phloem than they did on susceptible plants. They also showed that their feeding was frequently interrupted. Tests with [(14)C]sucrose showed that insects ingested much less phloem sap from the resistant than the susceptible plants. BPH feeding up-regulated callose synthase genes and induced callose deposition in the sieve tubes at the point where the stylet was inserted. The compact callose remained intact in the resistant plants, but genes encoding beta-1,3-glucanases were activated, causing unplugging of the sieve tube occlusions in susceptible plants. Continuing ingestion led to a remarkable reduction in the susceptible plants' sucrose content and activation of the RAmy3D gene, leading to starch hydrolysis and ultimately carbohydrate deprivation in the plants. Our results demonstrate that BPH feeding induces the deposition of callose on sieve plates in rice and that this is an important defense mechanism that prevents insects from ingesting phloem sap. In response, however, the BPH can unplug sieve tube occlusions by activating beta-1,3-glucanase genes in rice plants.