Effects of single and multiple nucleotide mutations on loop-mediated isothermal amplification.

Testing is pivotal for early identification of disease and subsequent infection control. Pathogens' nucleic acid sequence can change due to naturally-occurring genetic drift or intentional modification. Because of the reliance on molecular assays for human, animal, and plant disease diagnosis, we must understand how nucleotide mutations affect test accuracy. Primers designed against original lineages of a pathogen may be less efficient at detecting variants with genetic changes in priming regions. Here, we made single- and multi-point mutations in priming regions of a model SARS-CoV-2 template that was used as input for a loop-mediated isothermal amplification (LAMP) assay. We found that many of the modifications impacted assay sensitivity, amplification speed, or both. Further research exploring mutations at every position in each of the eight priming regions should be conducted to evaluate trends and determine generalizability.

Spodoptera frugiperda: Ecology, Evolution, and Management Options of an Invasive Species.

The fall armyworm (FAW), Spodoptera frugiperda (Lepidoptera, Noctuidae), is a well-known agricultural pest in its native range, North and South America, and has become a major invasive pest around the globe in the past decade. In this review, we provide an overview to update what is known about S. frugiperda in its native geographic ranges. This is followed by discussion of studies from the invaded areas to gain insights into S. frugiperda's ecology, specifically its reproductive biology, host plant use, status of insecticide resistance alleles, and biocontrol methods in native and invasive regions. We show that reference to host strains is uninformative in the invasive populations because multidirectional introduction events likely underpinned its recent rapid spread. Given that recent genomic analyses show that FAW is much more diverse than was previously assumed, and natural selection forces likely differ geographically, region-specific approaches will be needed to control this global pest.

Spodoptera frugiperda in Togo 5 years on: early impact of the invasion and future developments.

The infestation of the fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) in Africa since 2016 has been a major threat to maize production. Previous studies in Togo and Ghana from 2016 to 2018 did not correlate FAW infestation to yield losses. Thus, the aim of this study which assesses the impact of FAW infestation by inspecting 150 maize farms throughout the five Agro-Ecological Zones (AEZs) of Togo for FAW plant damage, and third instar larvae were used to infest 10-day-old maize plants in netted plots under controlled conditions at an experiment station (Station d'Expérimentations Agronomiques de Lomé) in 2019 and 2020. As control plots at the experiment station, plots were both netted and treated with emamectin benzoate, simply netted, or open to natural infestation. The number of larvae, egg masses, percent damaged plants, and damage proportions of leaves and ears were scored until harvest. Infestations and damages on maize plant throughout Togo were similar between the two years but were higher in the southern part of the county (AEZ5). At the experiment station, the yield losses were significantly considerable and increased from 25% infestation. The losses were 0.37 t ha-1 for 25% infestation, 0.34 t ha-1 for 30%, 0.59 t ha-1 for the open plots, 0.70 t ha-1 for simple netted and 50% infestation, 1.03 t ha-1 for 75%, and 1.27 t ha-1 for 100% infestation. This current study suggested thorough inspection on maize farms to set off management practices from 25% of infestation.

Whole genome comparisons reveal panmixia among fall armyworm (Spodoptera frugiperda) from diverse locations.

BACKGROUND: The fall armyworm (Spodoptera frugiperda (J.E. Smith)) is a highly polyphagous agricultural pest with long-distance migratory behavior threatening food security worldwide. This pest has a host range of > 80 plant species, but two host strains are recognized based on their association with corn (C-strain) or rice and smaller grasses (R-strain). The population genomics of the United States (USA) fall armyworm remains poorly characterized to date despite its agricultural threat. RESULTS: In this study, the population structure and genetic diversity in 55 S. frugiperda samples from Argentina, Brazil, Kenya, Puerto Rico and USA were surveyed to further our understanding of whole genome nuclear diversity. Comparisons at the genomic level suggest a panmictic S. frugiperda population, with only a minor reduction in gene flow between the two overwintering populations in the continental USA, also corresponding to distinct host strains at the mitochondrial level. Two maternal lines were detected from analysis of mitochondrial genomes. We found members from the Eastern Hemisphere interspersed within both continental USA overwintering subpopulations, suggesting multiple individuals were likely introduced to Africa. CONCLUSIONS: Our research is the largest diverse collection of United States S. frugiperda whole genome sequences characterized to date, covering eight continental states and a USA territory (Puerto Rico). The genomic resources presented provide foundational information to understand gene flow at the whole genome level among S. frugiperda populations. Based on the genomic similarities found between host strains and laboratory vs. field samples, our findings validate the experimental use of laboratory strains and the host strain differentiation based on mitochondria and sex-linked genetic markers extends to minor genome wide differences with some exceptions showing mixture between host strains is likely occurring in field populations.

A novel reference dated phylogeny for the genus Spodoptera Guenée (Lepidoptera: Noctuidae: Noctuinae): new insights into the evolution of a pest-rich genus.

The noctuid genus Spodoptera currently consists of 31 species with varied host plant breadths, ranging from monophagous and oligophagous non-pest species to polyphagous pests of economic importance. Several of these pest species have become major invaders, colonizing multiple continents outside their native range. Such is the case of the infamous fall armyworm, Spodoptera frugiperda (J.E. Smith), which includes two recognized host strains that have not been treated as separate species. Following its accidental introduction to Africa in 2016, it quickly spread through Africa and Asia to Australia. Given that half the described Spodoptera species cause major crop losses, comparative genomics studies of several Spodoptera species have highlighted major adaptive changes in genetic architecture, possibly relating to their pest status. Several recent population genomics studies conducted on two species enable a more refined understanding of their population structures, migration patterns and invasion processes. Despite growing interest in the genus, the taxonomic status of several Spodoptera species remains unstable and evolutionary studies suffer from the absence of a robust and comprehensive dated phylogenetic framework. We generated mitogenomic data for 14 Spodoptera taxa, which are combined with data from 15 noctuoid outgroups to generate a resolved mitogenomic backbone phylogeny using both concatenation and multi-species coalescent approaches. We combine this backbone with additional mitochondrial and nuclear data to improve our understanding of the evolutionary history of the genus. We also carry out comprehensive dating analyses, which implement three distinct calibration strategies based on either primary or secondary fossil calibrations. Our results provide an updated phylogenetic framework for 28 Spodoptera species, identifying two well-supported ecologically diverse clades that are recovered for the first time. Well-studied larvae in each of these clades are characterized by differences in mandibular shape, with one clade's being more specialized on silica-rich C4 grasses. Interestingly, the inferred timeframe for the genus suggests an earlier origin than previously thought for the genus: about 17-18 million years ago.

Stenotrophomonas maltophilia Differential Gene Expression in Synthetic Cystic Fibrosis Sputum Reveals Shared and Cystic Fibrosis Strain-Specific Responses to the Sputum Environment.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can infect the lungs of people with cystic fibrosis (CF). The highly viscous mucus in the CF lung, expectorated as sputum, serves as the primary nutrient source for microbes colonizing this site and induces virulence-associated phenotypes and gene expression in several CF pathogens. Here, we characterized the transcriptional responses of three S. maltophilia strains during exposure to synthetic CF sputum medium (SCFM2) to gain insight into how this organism interacts with the host in the CF lung. These efforts led to the identification of 881 transcripts differentially expressed by all three strains, many of which reflect the metabolic pathways used by S. maltophilia in sputum, as well as altered stress responses. The latter correlated with increased resistance to peroxide exposure after pregrowth in SCFM2 for two of the strains. We also compared the SCFM2 transcriptomes of two S. maltophilia CF isolates to that of the acute infection strain, S. maltophilia K279a, allowing us to identify CF isolate-specific signatures in differential gene expression. The expression of genes from the accessory genomes was also differentially altered in response to SCFM2. Finally, a number of biofilm-associated genes were differentially induced in SCFM2, particularly in K279a, which corresponded to increased aggregation and biofilm formation in this strain relative to both CF strains. Collectively, this work details the response of S. maltophilia to an environment that mimics important aspects of the CF lung, identifying potential survival strategies and metabolic pathways used by S. maltophilia during infections.IMPORTANCEStenotrophomonas maltophilia is an important infecting bacterium in the airways of people with cystic fibrosis (CF). However, compared to the other CF pathogens, S. maltophilia has been relatively understudied. The significance of our research is to provide insight into the global transcriptomic changes of S. maltophilia in response to a medium that was designed to mimic important aspects of the CF lung. This study elucidates the overall metabolic changes that occur when S. maltophilia encounters the CF lung and generates a road map of candidate genes to test using in vitro and in vivo models of CF.

Dual Gene Expression Analysis Identifies Factors Associated with Staphylococcus aureus Virulence in Diabetic Mice.

Staphylococcus aureus is a major human pathogen of the skin. The global burden of diabetes is high, with S. aureus being a major complication of diabetic wound infections. We investigated how the diabetic environment influences S. aureus skin infection and observed an increased susceptibility to infection in mouse models of both type I and type II diabetes. A dual gene expression approach was taken to investigate transcriptional alterations in both the host and bacterium after infection. While analysis of the host response revealed only minor changes between infected control and diabetic mice, we observed that S. aureus isolated from diabetic mice had significant increases in the levels of genes associated with translation and posttranslational modification and chaperones and reductions in the levels of genes associated with amino acid transport and metabolism. One family of genes upregulated in S. aureus isolated from diabetic lesions encoded the Clp proteases, associated with the misfolded protein response. The Clp proteases were found to be partially glucose regulated as well as influencing the hemolytic activity of S. aureus Strains lacking the Clp proteases ClpX, ClpC, and ClpP were significantly attenuated in our animal model of skin infection, with significant reductions observed in dermonecrosis and bacterial burden. In particular, mutations in clpP and clpX were significantly attenuated and remained attenuated in both normal and diabetic mice. Our data suggest that the diabetic environment also causes changes to occur in invading pathogens, and one of these virulence determinants is the Clp protease system.

Colorimetric-Luminance Readout for Quantitative Analysis of Fluorescence Signals with a Smartphone CMOS Sensor.

Smartphones have shown promise as an enabling technology for portable and distributed point-of-care diagnostic tests. The CMOS camera sensor can be used for detecting optical signals, including fluorescence for applications such as isothermal nucleic acid amplification tests. However, such analysis is typically limited mostly to end point detection of single targets. Here we present a smartphone-based image analysis pipeline that utilizes the CIE xyY (chromaticity-luminance) color space to measure the luminance (in lieu of RGB intensities) of fluorescent signals arising from nucleic acid amplification targets, with a discrimination sensitivity (ratio between the positive to negative signals), which is an order of magnitude more than traditional RGB intensity based analysis. Furthermore, the chromaticity part of the analysis enables reliable multiplexed detection of different targets labeled with spectrally separated fluorophores. We apply this chromaticity-luminance formulation to simultaneously detect Zika and chikungunya viral RNA via end point RT-LAMP (Reverse transcription Loop-Mediated isothermal amplification). We also show real time LAMP detection of Neisseria gonorrhoeae samples down to a copy number of 3.5 copies per 10 µL of reaction volume in our smartphone-operated portable LAMP box. Our chromaticity-luminance analysis is readily adaptable to other types of multiplexed fluorescence measurements using a smartphone camera.

Contrasting insect attraction and herbivore-induced plant volatile production in maize.

MAIN CONCLUSION: The maize inbred line W22 has lower herbivore-induced volatile production than B73 but both fall armyworm larvae and the wasps that parasitize them prefer W22 over B73. Maize inbred line W22 is an important resource for genetic studies due to the availability of the UniformMu mutant population and a complete genome sequence. In this study, we assessed the suitability of W22 as a model for tritrophic interactions between maize, Spodoptera frugiperda (fall armyworm) and the parasitoid wasp Cotesia marginiventris. W22 was found to be a good model for studying the interaction as S. frugiperda prefers W22 over B73 and a higher parasitism rate by C. marginiventris was observed on W22 compared to the inbred line B73. W22 also produced lower amounts of many herbivore-induced volatile terpenes and indole emission upon treatment with S. frugiperda oral secretions. We propose that some of the major herbivore-induced terpene volatiles are perhaps impeding S. frugiperda and C. marginiventris preference and that as yet unidentified compounds are produced at low abundance may be positively impacting these interactions.

Intraspecific differences in plant defense induction by fall armyworm strains.

The underlying adaptive mechanisms by which insect strains are associated with specific plants are largely unknown. In this study, we investigated the role of herbivore-induced defenses in the host plant association of fall armyworm (Spodoptera frugiperda) strains. We tested the expression of herbivore-induced defense-related genes and the activity of plant-defensive proteins in maize and Bermuda grass upon feeding by fall armyworm strains. The rice strain caterpillars induced greater accumulation of proteinase inhibitors in maize than the corn strain caterpillars. In Bermuda grass, feeding by the corn strain suppressed induction of trypsin inhibitor activity whereas the rice strain induced greater activity levels. Differences in elicitation of these plant defenses by the two strains seems to be due to differences in the activity levels of the salivary enzyme phospholipase C. The levels of plant defense responses were negatively correlated with caterpillar growth, indicating a fitness effect. Our results indicate that specific elicitors in the saliva of fall armyworm stains trigger differential levels of plant defense responses that affect caterpillar growth and thus may influence host plant associations in field conditions. The composition and secretion of plant defense elicitors may have a strong influence in the host plant association of insect herbivores.

Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA.

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer dimers and hairpins on previously published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter that can be correlated to the probability of non-specific amplification associated with LAMP primers.

Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses.

Reverse-transcription-loop-mediated isothermal amplification (RT-LAMP) has frequently been proposed as an enabling technology for simplified diagnostic tests for RNA viruses. However, common detection techniques used for LAMP and RT-LAMP have drawbacks, including poor discrimination capability, inability to multiplex targets, high rates of false positives, and (in some cases) the requirement of opening reaction tubes postamplification. Here, we present a simple technique that allows closed-tube, target-specific detection, based on inclusion of a dye-labeled primer that is incorporated into a target-specific amplicon if the target is present. A short, complementary quencher hybridizes to unincorporated primer upon cooling down at the end of the reaction, thereby quenching fluorescence of any unincorporated primer. Our technique, which we term QUASR (for quenching of unincorporated amplification signal reporters, read "quasar"), does not significantly reduce the amplification efficiency or sensitivity of RT-LAMP. Equipped with a simple LED excitation source and a colored plastic gel filter, the naked eye or a camera can easily discriminate between positive and negative QUASR reactions, which produce a difference in signal of approximately 10:1 without background subtraction. We demonstrate that QUASR detection is compatible with complex sample matrices such as human blood, using a novel LAMP primer set for bacteriophage MS2 (a model RNA virus particle). Furthermore, we demonstrate single-tube duplex detection of West Nile virus (WNV) and chikungunya virus (CHIKV) RNA.

F2 screen for resistance to Bacillus thuringiensis Cry2Ab2-maize in field populations of Spodoptera frugiperda (Lepidoptera: Noctuidae) from the southern United States.

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), is a target pest of transgenic maize and cotton expressing Bacillus thuringiensis (Bt) proteins in both North and South America. In 2013 and 2014, a total of 215 F2 two-parent families of S. frugiperda were established using single-pair mating of field individuals collected from seven locations in four states of the southern U.S.: Texas, Louisiana, Georgia, and Florida. The objective of the investigation was to detect resistance alleles in field populations to Cry2Ab2, a common Bt protein produced in transgenic maize and cotton. For each F2 family, 128 F2 neonates were screened on leaf tissue of Cry2Ab2 maize plants in the laboratory. A conservative estimate of the frequency of major Cry2Ab2 resistance alleles in S. frugiperda from the four states was 0.0023 with a 95% credibility interval of 0.0003-0.0064. In addition, six families were considered to likely possess minor resistance alleles at a frequency of 0.0082 with a 95% credibility interval of 0.0033-0.0152. One F2 family from Georgia (GA-15) was confirmed to possess a major resistance allele to the Cry2Ab2 protein. Larvae from this family survived well on whole maize plants expressing Cry2Ab2 protein and demonstrated a significant level (>15-fold) of resistance when fed with the same protein incorporated in a meridic diet. The detection of the major resistance allele along with the relatively abundant minor resistance alleles revealed in this study may have important implications for resistance management.

Transcriptomic Analysis of Yersinia enterocolitica Biovar 1B Infecting Murine Macrophages Reveals New Mechanisms of Extracellular and Intracellular Survival.

Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocolitica biovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26 °C to establish a baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37 °C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.

Demonstration Using Field Collections that Argentina Fall Armyworm Populations Exhibit Strain-specific Host Plant Preferences.

Spodoptera frugiperda, the fall armyworm, is a major economic pest throughout the Western Hemisphere of corn (maize), cotton, sorghum, and a variety of agricultural grasses and vegetable crops. Studies in the United States, the Caribbean, and Brazil demonstrated the existence of two subpopulations (previously designated "host strains") that differ in their choice of plant host. Specifically, the corn strain is preferentially found in corn and sorghum, while the rice strain is dominant in rice, turf grass, and alfalfa. However, inconsistent results were reported in surveys of fall armyworm in Argentina, with some indicating that the host plant preferences of the two strains might be compromised or even nonexistent. If correct, this would complicate efforts to control this pest by considerably expanding the range of habitats that would have to be considered as potential sources for fall armyworm infestations in specific crops. A reexamination of Argentine fall armyworm, this time with field collections rather than the laboratory colonies used in previous studies, confirmed the existence of the two strains and their host preferences. Specifically, the corn strain was consistently the majority population infesting corn and was usually so in sorghum, while the rice strain was predominant in pasture/turf grasses and alfalfa. The one outlier was a collection from rice, which had a corn strain majority. Overall, the data were generally consistent with strain behaviors observed in other areas of the Western Hemisphere.

Haplotype Profile Comparisons Between Spodoptera frugiperda (Lepidoptera: Noctuidae) Populations From Mexico With Those From Puerto Rico, South America, and the United States and Their Implications to Migratory Behavior.

Fall armyworm [Spodoptera frugiperda (J. E. Smith)] is a major economic pest throughout the Western Hemisphere of maize, cotton, sorghum, and a variety of agricultural grasses and vegetable crops. Previous studies demonstrated extensive annual migrations occurring as far north as Canada from overwintering locations in southern Florida and Texas. In contrast, migratory behavior in the rest of the hemisphere is largely uncharacterized. Understanding the migration patterns of fall armyworm will facilitate efforts to predict the spread of pesticide resistance traits that repeatedly arise in this species and assess the consequences of changing climatic trends on the infestation range. Four independent fall armyworm colonies derived from widely separated populations in Mexico and two field collections were examined for their mitochondrial cytochrome oxidase I (COI) gene haplotypes and compared with other locations. The Mexico populations were most similar in their haplotype profile to those from Texas and South America, but also displayed some distinctive features. The data extend the haplotype distribution map in the Western Hemisphere and confirm that the previously observed regional differences in haplotype frequencies are stable over time. The Mexico collections were associated with haplotypes rarely found elsewhere, suggesting limited migratory interactions with foreign populations, including those in neighboring Texas.

Assessing the resolution of haplotype distributions to delineate fall armyworm (Lepidoptera: Noctuidae) migratory behaviors.

Regions of southern Florida and southern Texas (extending into Mexico) provide the overwintering source populations for virtually all fall armyworm infestations affecting the continental United States. Understanding how these migratory populations annually disperse is important to predict and control infestations by this specific pest and to more generally investigate the environmental factors that influence the long-distance movements of flying insects. The two overwintering locations are associated with differences in the distribution of certain mitochondrial haplotypes that overlap in the region near the border separating the states of Alabama and Georgia. This provided an opportunity to test the resolution of the haplotype method by comparisons between smaller geographical areas and shorter time frames than previously examined. Correspondences were found between trap-capture numbers, fall armyworm strain proportions, and haplotype ratios calculated for individual counties and within season time periods that were generally consistent with expectations, providing confidence that those population movements could be accurately inferred. The comparison of haplotype distributions identified a migratory boundary separating the Texas and Florida populations coincident with the eastern edge of the Apalachicola-Chattahoochee-Flint River basin. Calculations of strain numbers based on genetic markers revealed similarities and differences in strain population dynamics that can be applied to study the migratory behavior of fall armyworm subpopulations. The use of this methodology for the detailed mapping of migratory pathways and the identification of factors that influence the direction and extent of pest migration are discussed.

Development of a Colorimetric Loop-Mediated Isothermal Amplification Assay for the Detection of Trypanosoma cruzi in Low-Resource Settings.

Chagas disease is an inflammatory parasitic infection caused by Trypanosoma cruzi (T. cruzi). Early diagnosis is crucial in guiding treatment and slowing disease progression; however, current diagnostic methods have insufficient detection limits and often require skilled technicians. Molecular tests, especially isothermal nucleic acid assays, are advantageous due to their excellent sensitivity, specificity, speed, and simplicity. Here, we optimized a colorimetric loop-mediated isothermal amplification (LAMP) assay for T. cruzi. We can detect as few as 2 genomic copies/reaction using three different T. cruzi strains. We examined selectivity using other parasitic protozoans and successfully detected T. cruzi DNA extracted from parasites in human whole blood down to 1.2 parasite equivalents/reaction. We also performed a blinded study using canine blood samples and established a 100% sensitivity, specificity, and accuracy for the colorimetric LAMP assay. Finally, we used a heated 3D printer bed and an insulated thermos cup to demonstrate that the LAMP incubation step could be performed with accessible, low-cost materials. Altogether, we have developed a high-performing assay for T. cruzi with a simple colorimetric output that would be ideal for rapid, low-cost screening at the point of use.

Pheromonal divergence between two strains of Spodoptera frugiperda.

Spodoptera frugiperda consists of two genetically and behaviorally different strains, the corn- and the rice-strain, which seem to be in the process of sympatric speciation. We investigated the role of strain-specific sexual communication as a prezygotic mating barrier between both strains by analyzing strain-specific variation in female pheromone composition of laboratory and field strains, and also male attraction in wind tunnel and field experiments. Laboratory-reared and field-collected females from Florida exhibited strain-specific differences in their relative amount of (Z)-7-dodecenyl acetate (Z7-12:OAc) and (Z)-9-dodecenyl acetate (Z9-12:OAc). In wind tunnel assays, we did not find strain-specific attraction of males to females. However, in field experiments in Florida, we observed some differential attraction to synthetic pheromone blends. In a corn field, the corn-strain blend attracted more males of both strains than the rice-strain blend, but both blends were equally attractive in a grass field. Thus, habitat-specific volatiles seemed to influence male attraction to pheromones. In dose-response experiments, corn-strain males were more attracted to 2 % Z7-12:OAc than other doses tested, while rice-strain males were attracted to a broader range of Z7-12:OAc (2-10 %). The attraction of corn-strain males to the lowest dose of Z7-12:OAc corresponds to the production of this compound by females; corn-strain females produced significantly smaller amounts of Z7-12:OAc than rice-strain females. Although corn-strain individuals are more restricted in their production of and response to pheromones than rice-strain individuals, it seems that differences in sexual communication between corn- and rice-strain individuals are not strong enough to cause assortative mating.

Advances in RT-LAMP for COVID-19 testing and diagnosis.

INTRODUCTION: The SARS-CoV-2 pandemic, and the subsequent limitations on standard diagnostics, has vastly expanded the user base of Reverse Transcription Loop-mediated isothermal Amplification (RT-LAMP) in fundamental research and development. RT-LAMP has also penetrated commercial markets, with emergency use authorizations for clinical diagnosis. AREAS COVERED: This review discusses the role of RT-LAMP within the context of other technologies like RT-qPCR and rapid antigen tests, progress in sample preparation strategies to enable simplified workflow for RT-LAMP directly from clinical specimens, new challenges with primer and assay design for the evolving pandemic, prominent detection modalities including colorimetric and CRISPR-mediated methods, and translational research and commercial development of RT-LAMP for clinical applications. EXPERT OPINION: RT-LAMP occupies a middle ground between RT-qPCR and rapid antigen tests. The simplicity approaches that of rapid antigen tests, making it suitable for point-of-care use, but the sensitivity nears that of RT-qPCR. RT-LAMP still lags RT-qPCR in fundamental understanding of the mechanism, and the interplay between sample preparation and assay performance. Industry is now beginning to address issues around scalability and usability, which could finally enable LAMP and RT-LAMP to find future widespread application as a diagnostic for other conditions, including other pathogens with pandemic potential.