A TaqMan qPCR method for detecting kdr resistance in Aphis gossypii demonstrates improved sensitivity compared to conventional PCR–RFLP

BackgroundAnopheles funestus, which is considered as secondary vector of malaria in Ethiopia, is known to have several morphologically indistinguishable (sibling) species. Accurate identification of sibling species is crucial to understand their biology, behaviour and vector competence. In this study, molecular identification was conducted on the Ethiopian An. funestus populations. Moreover, insecticide resistance mechanism markers were detected, including ace N485I, kdr L1014F, L1014S, and CYP6P9a TaqMan qPCR was used to detect the infective stage of the parasite from field collected adult female An. funestus populations.MethodsAdult female mosquito collection was conducted from Lare, Gambella Regional State of Ethiopia between June 2018 to July 2020 using CDC light traps and HLC. Sub-samples of the morphologically identified An. funestus mosquitoes were molecularly identified using species-specific PCR, and the possible presence of insecticide resistance alleles was investigated using TaqMan qPCR (N485I-Ace-1), PCR-Sanger sequencing (L1014F-kdr), and PCR–RFLP (CYP6P9a resistance allele). Following head/thorax dissection, the TaqMan qPCR assay was used to investigate the presence of the infective stage Plasmodium parasite species.ResultsA total of 1086 adult female An. funestus mosquitoes were collected during the study period. All sub-samples (N = 20) that were morphologically identified as An. funestus sensu lato (s.l.) were identified as An. funestus sensu stricto (s.s.) using species- specific PCR assay. The PCR–RFLP assay that detects the CYP6P9a resistance allele that confers pyrethroid resistance in An. funestus was applied in N = 30 randomly selected An. funestus s.l. specimens. None of the specimens showed a digestion pattern consistent with the presence of the CYP6P9a resistance allele in contrast to what was observed in the positive control. Consequently, all samples were characterized as wild type. The qPCR TaqMan assay that detects the N485I acetylcholinesterase-1 mutation conferring resistance to organophosphates/carbamates in An. funestus was used in (N = 144) samples. All samples were characterized as wild type. The kdr L1014F and L1014S mutations in the VGSC gene that confer resistance to pyrethroids and DDT were analysed with direct Sanger sequencing after PCR and clean-up of the PCR products were also characterized as wild type. None of the samples (N = 169) were found positive for Plasmodium (P. falciparum/ovale/malariae/vivax) detection.ConclusionAll An. funestus s.l. samples from Lare were molecularly identified as An. funestus s.s. No CYP6P9, N485I acetylcholinesterase 1, kdr L1014F or L1014S mutations were detected in the An. funestus samples. None of the An. funestus samples were positive for Plasmodium. Although the current study did not detect any insecticide resistant mechanism, it provides a reference for future vector monitoring programmes. Regular monitoring of resistance mechanisms covering wider geographical areas of Ethiopia where this vector is distributed is important for improving the efficacy of vector control programs.

This study explored the applicability of using TaqMan qPCR (quantitative polymerase chain reaction) for Escherichia coli, Salmonella enterica and non-virulent Vibrio cholerae detection in surface and drinking water. One hundred and twenty water samples were collected monthly (January 2017–December 2017) from the surface water (input) and drinking water (output and distribution networks) of two drinking water treatment plants (DWTPs) in Damietta County, Egypt. The distribution of the studied bacteria based on their detection by TaqMan qPCR compared with membrane filtration (MF) technique showed that the higher positive samples were detected by TaqMan qPCR. The bacterial count was totally absent in all output samples. TaqMan qPCR assay (based on sequence detection of uidA, invA, and ompW) revealed 97.96%, 99.14%, and 98.3% specificity for E. coli, S. enterica, and non-virulent V. cholerae, respectively, compared with 100% specificity for all strains when MF cultures were applied. TaqMan qPCR exhibited 100% sensitivity for all strains, while it was 91.67%, 80%, and 50% using MF cultures for E. coli, S. enterica, and non-virulent V. cholerae, respectively. In conclusion, TaqMan qPCR sensitivity makes it a useful tool for urgent fast monitoring of water contamination, especially in network samples that contain low bacterial count.

Studies on the natural factors contributing to pest regulation are fundamental to developing efficient integrated pest management programs. Chemical control is the main management method used for pests [e.g., Aphis gossypii (Glover)]. The studies of pest management with chemical control provide information that can be incorporated into integrated pest management programs to promote more sustainable pest control approaches. Here, we report the critical stages of A. gossypii and its abiotic and biotic natural mortality factors in cotton crops as a function of plant phenology using a life table. The critical stages of A. gossypii were the first and fourth instars. Together, the abiotic and biotic factors caused 94.31% of the mortality in the A. gossypii populations in cotton crops with plants in the vegetative, flowering, and fruiting stages. The key mortality factors were rainfall and predation. Syrphidae Allograpta exotica (Wiedemann) (Diptera: Syrphidae) and Chrysopidae Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) larvae, many Coccinellidae species Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae), Eriopsis connexa (Germar) (Coleoptera: Coccinellidae), Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), Hippodamia convergens (Guérin-Meneville) (Coleoptera: Coccinellidae), Scymnus rubicundus (Erichson) (Coleoptera: Coccinellidae) and Stethorus punctillum (Weise) (Coleoptera: Coccinellidae), one Anthocoridae species Orius insidiosus (Say) (Hemiptera: Anthocoridae), and individuals from the Araneidae family were responsible for the predation of A. gossypii. The results obtained in this study provide support for the idea that efforts to preserve natural enemies (e.g., predators) and rainfall monitoring should be adapted due to their importance for the regulation of A. gossypii populations in all the phenological stages of cotton in tropical regions.

The cotton aphid, Aphis gossypii (Glover), is a destructive pest that affects numerous crops throughout the world. Pyrethroid resistance has become endemic in A. gossypii populations in the cotton growing areas of China due to wide- spread application of insecticides. To assess the extent of pyrethroid resistance, bioassays were conducted on field populations collected from several cotton cultivation areas from 2010 to 2015. The frequency of a known resistance-associated sodium channel mutation (M918 L) in A. gossypii was evaluated and the bioassay of bifenthrin with or without the synergist was performed to illuminate the mechanisms underlying resistance to pyrethroids. The field populations exhibited very high levels of resistance to both beta-cypermethrin and deltamethrin. Pretreatment with synergists, DEF and PBO, significantly increased the toxicity of bifenthrin to cotton aphid populations collected from Bt cotton fields in China. Further, 96.8-100% of individuals with the M918 L mutation (including both RR and RS individuals) were observed in various populations, and only 2.8-3.2% of individuals with wild-type homozygotes (SS) were detected. The mutation M918 L in the voltage-gated sodium channel along with detoxifying metabolism was contributed to the pyrethroid resistance in the field populations of Aphis gossypii from cotton growing regions of China. And insecticides with different modes of action should be recommended for the control of A. gossypii in the future. © 2017 Society of Chemical Industry.

This study aims to provide a comprehensive understanding of the pest and natural enemy complex associated with okra during Kharif seasons. Okra (Abelmoschus esculentus L. Moench), an important vegetable crop in tropical and subtropical regions, is susceptible to a variety of pests that can significantly impact yield and quality. Pest management in okra is becoming increasingly challenging due to climate change and other environmental factors. These natural enemies include various species of ladybirds, spiders, mantises, and parasitoid wasps, which help regulate pest numbers and reduce the need for chemical pesticides, thereby promoting sustainable pest management practices. The study investigates the pest complex and their natural enemies in okra during the Kharif seasons of 2020-21 and 2022-23, aiming to provide insights into pest dynamics and inform integrated pest management (IPM) strategies. The major pests identified include sap-suckers (Jassids, Cotton aphids, Whiteflies, and Mites), defoliators (Red pumpkin beetles, Grasshoppers, and caterpillars), and borers (Blister beetles, fruit borers, and shoot borers), which cause significant damage from the vegetative to maturity stages of the crop. Natural enemies such as Coccinellids, spiders, spraying mantises, and parasitoid wasps play a critical role in regulating pest populations and enhancing biological control. Additionally, pollinators like honeybees contribute to fruit set and overall plant health. The study highlights the seasonal fluctuations of pest populations, the effectiveness of natural enemies, and the potential for sustainable pest management through biological control. Findings from this research will support the development of eco-friendly pest management practices, reducing the reliance on chemical pesticides and promoting a balanced agroecosystem for sustainable okra production.

A hemizygous 1.5-3.0-Mb microdeletion of human chromosome 22q11.2 with the loss of multiple genes including histone cell cycle regulator (HIRA) causes 22q11.2 deletion syndrome (22q11.2 DS), a common disorder with variable manifestations including congenital malformations affecting the heart, palate and kidneys in association with neurodevelopmental, psychiatric, endocrine and autoimmune abnormalities. The aim of this study was to develop a TaqMan-based dosage analysis PCR (TaqMan qPCR) for use as a rapid, cost-effective test for clinically suspected patients fulfilling previously described criteria for molecular diagnosis of 22q11.2 DS in a lower middle-income country where the cost of testing limits its use in routine clinical practice. Nineteen patients were recruited with informed consent following ethical approval from the Ethics Review Committee, Lady Ridgeway Hospital for Children, Colombo. Dosage analysis of extracted DNA was performed using a TaqMan qPCR assay by amplifying regions within the target (HIRA) and control (testin LIM domain protein (TES)) genes of suspected patient (P) and unaffected person (N) samples. For detection of a deletion, the normalized value (HIRA/TES dosage) of a P sample was compared with that of an N sample. A ratio of P:N of 0.5 confirmed the presence of a deletion while a ratio of 1.0 refuted this. Seven of the 19 patients were found to have a HIRA deletion, confirming the diagnosis of 22q11.2 DS, with these results being in complete agreement with those of fluorescence in situ hybridization (FISH) (performed in nine of the 19 cases) and whole-exome sequencing (all 19 samples tested). This TaqMan qPCR assay was able to reliably distinguish HIRA-deleted cases from non-deleted ones. The assay was both cheaper and faster compared to commercially available alternatives in our setting, including FISH and multiple ligation-dependent probe amplification.

Bordetella avium (BA) is one of many pathogens that cause respiratory diseases in turkeys. However, other bacterial species can easily overgrow it during isolation attempts. This makes confirming the diagnosis of BA as the causative agent of turkey coryza more difficult. Currently, there are two PCR assays for the molecular detection of BA. One is conventional gel-based PCR and the other is TaqMan real-time PCR (qPCR) assay. However, multiple pitfalls were detected in both assays regarding their specificity, sensitivity, and efficiency, which limits their utility as diagnostic tools. In this study, we developed and validated two TaqMan qPCR assays and compared their performance to the currently available TaqMan qPCR. The two assays were able to correctly identify all BA isolates and showed negative results against a wide range of different microorganisms. The two assays were found to have high efficiency with a detection limit of approximately 1 × 103 plasmid DNA Copies/mL with high repeatability and reproducibility. In comparison to the currently available TaqMan qPCR assay, the newly developed assays showed significantly higher PCR efficiencies due to superior primers and probes design. The new assays can serve as a reliable tool for the sensitive, specific, and efficient diagnosis of BA.

Bacillus subtilis, a typical plant growth-promoting rhizobacteria, can benefit plant through promoting growth and reducing disease. The colonization intensity of B. subtilis in rhizosphere is a key factor for improving their effectiveness of field application. In this study, we developed a rapid and sensitive method for detecting B. subtilis in rhizosphere via TaqMan qPCR and droplet digital PCR (ddPCR) methods. The primers/probe set targeting gyrB gene could successfully distinguish B. subtilis from its close-related species. Both the TaqMan qPCR and ddPCR methods exhibited a good linear relationship in the sensitivity assay, suggesting the developed method was specific, effective and reliable. Finally, the two methods were used to detect the colonization dynamic of B. subtilis within Arabidopsis rhizosphere. Both of them showed a consistent trend compared with the traditional cultivation-based and microscopy-based methods. The TaqMan qPCR and droplet digital PCR (ddPCR) methods we developed could be used to rapidly detect B. subtilis in rhizosphere. The TaqMan qPCR and ddPCR methods developed in this study can be applied to rapid quantitative detection of B. subtilis populations, and will be helpful to evaluate their effectiveness of field application.

Triacylglycerols (TAGs) are the primary form of energy storage in eukaryotes. They also serve as a reservoir of fatty acids for membrane biogenesis of the cells and lead to obesity when excessively accumulated in adipose tissues. Diacylglycerol acyltransferases (DGATs) are responsible for the final and rate-limiting step of TAG biosynthesis in eukaryotic organisms. Understanding the roles of DGATs will help to create transgenic plants and microbes with value-added properties and provide information for therapeutic intervention for obesity and related diseases. Mammalian DGATs are divided into DGAT1 and DGAT2 subfamilies. It was unclear which DGAT was the major isoform expressed in animals. The objective of this study was to identify the major form of DGATs expressed in animal cells and compared to those expressed in tung tree seeds. TaqMan and SYBR Green qPCR assays evaluated DGAT mRNA levels in mouse 3T3-L1 adipocytes and RAW264.7 macrophages and tung tree seeds. TaqMan qPCR showed that DGAT mRNA levels were stable during the time-course study, DGAT2 mRNA levels were 10–30 folds higher than DGAT1 in adipocytes and macrophages, and DGAT mRNA levels in adipocytes were 50–100 folds higher than those in macrophages. As a positive control, the anti-inflammatory tristetraprolin (TTP) mRNA levels were 2–4 folds higher in macrophages than those in adipocytes and similar to DGAT1 in adipocytes but 100-fold higher than DGAT1 in macrophages. SYBR Green qPCR analyses confirmed TaqMan qPCR results. DGAT2 mRNA as the major DGAT mRNA in mouse cells was similar to that in tung tree seeds where DGAT2 mRNA levels were 10–20-fold higher than DGAT1 or DGAT3. Our results conclusively demonstrated that DGAT2 mRNA was the major form of DGATs expressed in plant and animal cells. This finding should facilitate efforts to regulate lipid/oil accumulation in plants and animals. Support or Funding Information Supported by USDA-ARS Quality and Utilization of Agricultural Products Research Program 306 through CRIS 6054-41000-103-00D. DGAT2 mRNA as the major form in eukaryote cells This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Aphis gossypii, one of the most important agricultural pests in the world, can cause serious economic losses in the main crop-producing areas. To clarify issues such as the genetic differentiation, genetic structure, and demographic history of A. gossypii populations, we used 10 nuclear microsatellite loci (SSR) and two mitochondrial gene sequences (COI and Cytb) to investigate genetic diversity and population structure of A. gossypii populations that were collected from 33 sampling sites in China from different climatic zones. SSR and mtDNA data suggested low to moderate levels of genetic diversity. A star-shaped network of mtDNA haplotypes indicated that the maternal ancestor of China cotton aphids likely originated in Xinjiang. The POPTREE, STRUCTURE and principal coordinate analysis (PCoA) revealed two genetic clusters: an eastern and a western region group. Isolation by distance (IBD) results showed a positive correlation between geographic distance and genetic distance in the vast eastern region but not in the western region. Neutrality testing and mismatch distribution analysis provided strong evidence for a recent rapid expansion in most populations. Genetic bottleneck was not detected in A. gossypii populations of China. The present work can help us to develop strategies for managing this pest.

BackgroundWith the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping.MethodsWhole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR.ResultsIn whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected.ConclusionsThese results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

BACKGROUND. With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. METHODS. Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 μL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. RESULTS. In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. CONCLUSIONS. These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

Widespread reports of reduced efficacy of imidacloprid for managing cotton aphids (Aphis gossypii Glover) in cotton (Gossypium hirsutum L.) prompted an investigation to characterize the susceptibility of 43 populations over a 2-year period. The susceptibility of A. gossypii populations to imidacloprid was examined by calculating LC50 values. Further analyses related resistance assay results to a gradient of cotton production intensity. Concentration-mortality bioassays documencted populations that were 4.26-607.16 times more resistant than the susceptible laboratory population. There was a significant positive relationship between LC50 values and percentage of cotton within 2.5- and 5-km buffers surrounding collection sites. No significant relationship was detected between LC50 values and the percentage of alternative crop and noncrop hosts. Variable and high levels of resistance were detected in A. gossypii populations, and this variation was positively associated with cotton production intensity. Cotton is a host that may receive multiple applications of neonicotinoids (via seed treatment and foliar sprays) annually for seedling and mid-season pests. Rotating modes of action and limiting insecticide use should be implemented to delay the evolution of insecticide resistance in A. gossypii populations. © 2022 Society of Chemical Industry.

Insecticide resistance in pepper greenhouse populations of Aphis gossypii (Hemiptera: Aphididae) in Korea

Australia is known worldwide for producing cotton of the finest quality. Cotton production has an important position in the economy of Australia, earning foreign exchange of two billion dollars annually. Australian cotton production is highly mechanized, and its production systems are quite specialized with emphasis on more crops per drop. However, several factors are making Australian cotton production challengeable, such as climate change, water scarcity, emergence of new insects, diseases and weeds, waterlogged soils and rising production cost. Australian cotton growers, as well as cotton scientists, put sincere efforts to cope up with these issues. New agronomic tools, for example, the skip‐row technique for water saving in the dry land area, timely planting, and use of glyphosate‐tolerant and Helicoverpa ‐resistant cultivars, were adopted to increase the yield potential. Synergy among weather forecasting, fertilizer, water, and pest management in the integrated crop management mode is being considered for high efficiency of cotton production systems. This book chapter explores current issues of Australian cotton production systems and opportunities to solve these issues.