Center for Grain and Animal Health Research

Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cell–cell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control. The red flour beetle Tribolium castaneum is a common pest: a type of 'bran bug', it targets cereal products, including grain, flour and rice bran. It is also a commonly used laboratory model, combining the ease of systematic RNA interference experiments such as those used with the nematode worm C. elegans with a biology that is more representative of most insects than even Drosophila. This weeks sees the publication by the Tribolium Genome Sequencing Consortium of the genomic sequence of T. castaneum. This is the first beetle genome to be published, and it will be a valuable resource for insect development studies and pest biology. The beetle Tribolium castaneum is a commonly used laboratory model, combining the ease of systematic RNAi experiments like those in Caenorhabditis elegans, with biology that is more representative of most insects than Drosophila melanogaster. A large consortium has sequenced and analysed the genome of the red flour beetle, creating a resource for biologists everywhere.

This review covers selected literature from 1982 to the present on some of the ecological, behavioral, and biochemical aspects of hydrocarbon use by insects and other arthropods. Major ecological and behavioral topics are species- and gender-recognition, nestmate recognition, task-specific cues, dominance and fertility cues, chemical mimicry, and primer pheromones. Major biochemical topics include chain length regulation, mechanism of hydrocarbon formation, timing of hydrocarbon synthesis and transport, and biosynthesis of volatile hydrocarbon pheromones of Lepidoptera and Coleoptera. In addition, a section is devoted to future research needs in this rapidly growing area of science.

Abstract Opal phytoliths derived from epidermal cells of grass leaves have been identified in atmospheric dust, soils, paleosols, Pleistocene loess, and deep‐sea sediments. By comparing oriented shapes of phytoliths in spodograms of 17 common grass species, four classes and 26 types are proposed which distinguish three groups of subfamilies of Gramineae. The Festucoid class contains eight types that are circular, rectangular, elliptical, or oblong forms. The Chloridoid class contains two types of saddle‐shaped bodies. The Panicoid class contains 11 types that are variations of crosses and dumbbells. The Elongate class contains five types that have no subfamily implications and occur in all 17 species. Because phytoliths of native tall grasses (Panicoid), short grasses (Chloridoid), and common domestic grasses of the humid regions (Festucoid) can be distinguished, it is possible to determine whether phytoliths in dust, soils, and sediments were derived from local or remote sources.

Fungal disease meets its match Fusarium head blight (FHB), caused by a fungus, reduces wheat crop yield and introduces toxins into the harvest. From the assembly of the genome of Thinopyrum elongatum , a wild relative of wheat used in breeding programs to improve cultivated wheat, Wang et al. cloned a gene that can address both problems (see the Perspective by Wulff and Jones). The encoded glutathione S -transferase detoxifies the trichothecene toxin and, when expressed in wheat, confers resistance to FHB. Science , this issue p. eaba5435 ; see also p. 822

Stored-product insects can cause postharvest losses, estimated from up to 9% in developed countries to 20% or more in developing countries. There is much interest in alternatives to conventional insecticides for controlling stored-product insects because of insecticide loss due to regulatory action and insect resistance, and because of increasing consumer demand for product that is free of insects and insecticide residues. Sanitation is perhaps the first line of defense for grain stored at farms or elevators and for food-processing and warehouse facilities. Some of the most promising biorational management tools for farm-stored grain are temperature management and use of natural enemies. New tools for computer-assisted decision-making and insect sampling at grain elevators appear most promising. Processing facilities and warehouses usually rely on trap captures for decision-making, a process that needs further research to optimize.

The order Coleoptera (beetles) is arguably the most speciose group of animals, but the evolutionary history of beetles, including the impacts of plant feeding (herbivory) on beetle diversification, remain poorly understood. We inferred the phylogeny of beetles using 4,818 genes for 146 species, estimated timing and rates of beetle diversification using 89 genes for 521 species representing all major lineages and traced the evolution of beetle genes enabling symbiont-independent digestion of lignocellulose using 154 genomes or transcriptomes. Phylogenomic analyses of these uniquely comprehensive datasets resolved previously controversial beetle relationships, dated the origin of Coleoptera to the Carboniferous, and supported the codiversification of beetles and angiosperms. Moreover, plant cell wall-degrading enzymes (PCWDEs) obtained from bacteria and fungi via horizontal gene transfers may have been key to the Mesozoic diversification of herbivorous beetles-remarkably, both major independent origins of specialized herbivory in beetles coincide with the first appearances of an arsenal of PCWDEs encoded in their genomes. Furthermore, corresponding (Jurassic) diversification rate increases suggest that these novel genes triggered adaptive radiations that resulted in nearly half of all living beetle species. We propose that PCWDEs enabled efficient digestion of plant tissues, including lignocellulose in cell walls, facilitating the evolution of uniquely specialized plant-feeding habits, such as leaf mining and stem and wood boring. Beetle diversity thus appears to have resulted from multiple factors, including low extinction rates over a long evolutionary history, codiversification with angiosperms, and adaptive radiations of specialized herbivorous beetles following convergent horizontal transfers of microbial genes encoding PCWDEs.

BACKGROUND: Sorghum possesses phenolic compounds that are health-promoting constituents of the grain. There are approximately 40 000 sorghum accessions, many of which have not been evaluated for the grain's health-promoting potential. Conventional methods for measuring total phenolic content, flavonoid content and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-scavenging capacity are time-consuming and labour-intensive, resulting in low overall throughput. The objective of this study was to develop a high-throughput screening assay for large sorghum sample sets to determine flavonoid and phenolic content and to modify existing DPPH and total phenolic assays. RESULTS: The 96-well assays exhibited a correlation of > 0.9 with the conventional assays. The 96-well assays allowed for up to 64 samples to be run per day compared with 20-24 samples (depending on the test) for the conventional methods. The 96-well assays had excellent accuracy (97.65-106.16% recovery), precision (1.06-8.28% coefficient of variation (CV)) and reproducibility (1.32-2.13% CV inter-day and 1.36-2.09% CV intra-day). CONCLUSION: The high-throughput 96-well plate method proved to be as robust and reproducible as the conventional method for determining total phenolic content, flavonoid content and DPPH-scavenging capacity in either sorghum bran or flour.

Drought and heat stress are among the two most important environmental factors influencing crop growth, development, and yield processes. A comprehensive understanding of the impact of drought and heat stress will be critical in evaluating the impact of climate change and climate variability on crop production. Both drought and heat stress influence an array of processes including physiological, growth, developmental, yield, and quality of crop. The objective of this review is to provide an overview of the influences of these two stresses on the above processes independently and in combination. Our review suggests a clear need of information on interactive effects of stresses particularly of drought and heat stress which mostly occur in combination. Both short- and long-term stresses can significantly influence growth and yield processes when stress occurs at sensitive stages. Crops are generally more sensitive to drought and/or heat stress during reproductive stages of development, which mainly influences seed numbers. Some of the important traits associated with drought- and/or heat-stress tolerance are indicated and discussed. The impacts of drought and heat stress are often different, and tolerance mechanisms may also be different. There is a wide range of crop modeling approaches (simple empirical models and more mechanistic models) that try to quantify the impact of stresses on growth, development, and yield and yield quality traits. These crop models should have the capability to quantify the impact of both short- and long-term stress events on growth, development, and yield processes. Modeling growth, development, sink-source relation, grain yield, and grain quality of crops can improve understanding of physiological and genetic nature of tolerance which can lead to increased grain yield and quality of crops. Improved models can enhance our capacity to predict crop performance in future climates and also to identify traits that can potentially be improved or exploited to obtain higher and more stable crop yields under stressed environments.

In conventional fault-tree analysis, the failure probabilities of components of a system are treated as exact values in estimating the failure probability of the top event. For many systems, it is often difficult to evaluate the failure probabilities of components from past occurrences because the environments of the systems change. Furthermore, it might be necessary to consider possible failure of components even if they have never failed before. We, therefore, propose to employ the possibility of failure, viz. a fuzzy set defined in probability space. The notion of the possibility of failure is more predictive than that of the probability of failure; the latter is a limiting case of the former. In the present approach based on a fuzzy fault-tree model, the maximum possibility of system failure is determined from the possibility of failure of each component within the system according to the extension principle. In calculating the possibility of system failure, some approximation is made for simplicity.

Drought tolerance is a key trait for increasing and stabilizing barley productivity in dry areas worldwide. Identification of the genes responsible for drought tolerance in barley (Hordeum vulgare L.) will facilitate understanding of the molecular mechanisms of drought tolerance, and also facilitate the genetic improvement of barley through marker-assisted selection or gene transformation. To monitor the changes in gene expression at the transcriptional level in barley leaves during the reproductive stage under drought conditions, the 22K Affymetrix Barley 1 microarray was used to screen two drought-tolerant barley genotypes, Martin and Hordeum spontaneum 41-1 (HS41-1), and one drought-sensitive genotype Moroc9-75. Seventeen genes were expressed exclusively in the two drought-tolerant genotypes under drought stress, and their encoded proteins may play significant roles in enhancing drought tolerance through controlling stomatal closure via carbon metabolism (NADP malic enzyme, NADP-ME, and pyruvate dehydrogenase, PDH), synthesizing the osmoprotectant glycine-betaine (C-4 sterol methyl oxidase, CSMO), generating protectants against reactive-oxygen-species scavenging (aldehyde dehydrogenase,ALDH, ascorbate-dependent oxidoreductase, ADOR), and stabilizing membranes and proteins (heat-shock protein 17.8, HSP17.8, and dehydrin 3, DHN3). Moreover, 17 genes were abundantly expressed in Martin and HS41-1 compared with Moroc9-75 under both drought and control conditions. These genes were possibly constitutively expressed in drought-tolerant genotypes. Among them, seven known annotated genes might enhance drought tolerance through signalling [such as calcium-dependent protein kinase (CDPK) and membrane steroid binding protein (MSBP)], anti-senescence (G2 pea dark accumulated protein, GDA2), and detoxification (glutathione S-transferase, GST) pathways. In addition, 18 genes, including those encoding Delta(l)-pyrroline-5-carboxylate synthetase (P5CS), protein phosphatase 2C-like protein (PP2C), and several chaperones, were differentially expressed in all genotypes under drought; thus they were more likely to be general drought-responsive genes in barley. These results could provide new insights into further understanding of drought-tolerance mechanisms in barley.

Background: The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem.\nResults: To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads.\nConclusions: Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

Outbreaks of monkeypox (mpox) have historically resulted from zoonotic spillover of clade I monkeypox virus (MPXV) in Central Africa and clade II MPXV in West Africa. In 2022, subclade IIb caused a global epidemic linked to transmission through sexual contact. Here we describe the epidemiological and genomic features of an mpox outbreak in a mining region in eastern Democratic Republic of the Congo, caused by clade I MPXV. Surveillance data collected between September 2023 and January 2024 identified 241 suspected cases. Genomic analysis demonstrates a distinct clade I lineage divergent from previously circulating strains in the Democratic Republic of the Congo. Of the 108 polymerase chain reaction-confirmed mpox cases, the median age of individuals was 22 years, 51.9% were female and 29% were sex workers, suggesting a potential role for sexual transmission. The predominance of APOBEC3-type mutations and the estimated emergence time around mid-September 2023 imply recent sustained human-to-human transmission.

Journal Article Insect Chemical Ecology: an Evolutionary Approach Get access Insect Chemical Ecology: An Evolutionary ApproachRoitberg B. D. Isman M. B. [eds.] Routledge, Chapman & Hall, New York, 1992 359 pp., $35.00 ISBN 0-412-01881-0 Ralph W. Howard Ralph W. Howard USDA-ARS Manhattan, KS. Search for other works by this author on: Oxford Academic Google Scholar Annals of the Entomological Society of America, Volume 86, Issue 4, 1 July 1993, Pages 507–508, https://doi.org/10.1093/aesa/86.4.507 Published: 01 July 1993

Functional analysis of the two chitin synthase genes, TcCHS1 and TcCHS2, in the red flour beetle, Tribolium castaneum, revealed unique and complementary roles for each gene. TcCHS1-specific RNA interference (RNAi) disrupted all three types of moult (larval-larval, larval-pupal and pupal-adult) and greatly reduced whole-body chitin content. Exon-specific RNAi showed that splice variant 8a of TcCHS1 was required for both the larval-pupal and pupal-adult moults, whereas splice variant 8b was required only for the latter. TcCHS2-specific RNAi had no effect on metamorphosis or on total body chitin content. However, RNAi-mediated down-regulation of TcCHS2, but not TcCHS1, led to cessation of feeding, a dramatic shrinkage in larval size and reduced chitin content in the midgut.

Two Bacillus thuringiensis(Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs. Two Bacillus thuringiensis(Bt)-resistant strains of the Indianmeal moth, Plodia interpunctella, lack a major gut proteinase that activates Bt protoxins. The absence of this enzyme is genetically linked to larval survival on Bt-treated diets. When considered with previous data supporting the existence of receptor-mediated insect resistance to Bt, these results provide evidence that insect adaptation to these toxins occurs through multiple physiological mechanisms, which complicate efforts to prevent or manage resistance to Bt toxins in insect control programs. Insecticidal proteins of the bacterium Bacillus thuringiensis (Bt) 1The abbreviations used are: Bt, Bacillus thuringiensis; BApNA,N-α-benzoyl-l-argininep-nitroanilide; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: Bt, Bacillus thuringiensis; BApNA,N-α-benzoyl-l-argininep-nitroanilide; PAGE, polyacrylamide gel electrophoresis. are effective for controlling many insect pest species, but insect resistance threatens the long term effectiveness of these toxins (1McGaughey W.H. Whalon M.E. Science. 1992; 258: 1451-1455Crossref PubMed Scopus (351) Google Scholar). With the introduction of transgenic plants expressing Bt toxins in the field (2Estruch J.J. Carozzi N.B. Desai N. Duck N.B. Warren G.W. Koziel M.G. Nature Biotechnol. 1997; 15: 137-141Crossref PubMed Scopus (194) Google Scholar), insects will likely be under increased selection pressure for resistance. To develop effective toxin resistance management strategies, a full understanding of the physiological and genetic mechanisms by which insects become resistant to these insecticidal proteins is needed. More than 100 insecticidal crystal protein genes from Bt subspecies have been described. 2For an up to date list and explanation of nomenclature, see:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/toxins.html. 2For an up to date list and explanation of nomenclature, see:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/toxins.html. These genes encode protoxins, referred to as Cry proteins, that have different specificity for lepidopteran, coleopteran, or dipteran insects. The Cry1 subclass consists of lepidopteran-active Bt protoxins with apparent molecular masses of approximately 130 kDa, which are solubilized and processed (activated) by gut enzymes to approximately 65-kDa toxins (4Höfte H. Whiteley H.R. Microbiol. Rev. 1989; 53: 242-255Crossref PubMed Google Scholar). Toxins interact with receptors in the guts of susceptible insects resulting in pore formation in midgut cell membranes, ionic imbalance, and consequent septicemia in the insect. Alterations in insect gut physiology or biochemistry could disrupt this sequential process and result in toxin resistance. Several mechanisms of insect resistance to Bt toxins have been proposed (5Gill S.S. Cowles E.A. Pietrantonio P.V. Annu. Rev. Entomol. 1992; 37: 615-636Crossref PubMed Google Scholar). One involves changes in the binding of toxins to gut receptors. In a Bt subspecies kurstaki-resistant strain of the Indianmeal moth, Plodia interpunctella, reduced binding of Cry1Ab toxin to larval brush border membrane vesicles was associated with increased resistance to the toxin (6van Rie J. McGaughey W.H. Johnson D.E. Barnett B.D. van Mellaert H. Science. 1990; 247: 72-74Crossref PubMed Scopus (285) Google Scholar). Decreases in toxin binding have also been reported in resistant strains of the diamondback moth, Plutella xylostella (7Ferré J. Real M.D. van Rie J. Jansens S. Perferoen M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5119-5123Crossref PubMed Scopus (300) Google Scholar, 8Bravo A. Hendrickx K. Jansens S. Peferoen M. J. Invertebr. Pathol. 1992; 60: 247-253Crossref Scopus (93) Google Scholar, 9Tabashnik B.E. Finson N. Groeters F.R. Moar W.J. Johnson M.W. Luo K. Adang M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4120-4124Crossref PubMed Scopus (198) Google Scholar, 10Masson L. Mazza A. Brousseau R. Tabashnik B. J. Biol. Chem. 1995; 270: 1-10Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 11Eschriche B. Tabashnik B. Finson N. Ferré J. Biochem. Biophys. Res. Commun. 1995; 212: 388-395Crossref PubMed Scopus (30) Google Scholar, 12Tang J.D. Shelton A.M. van Rie J. de Roeck S. Moar W.J. Roush R.T. Peferoen M. Appl. Environ. Microbiol. 1996; 62: 564-569Crossref PubMed Google Scholar), tobacco budworm, Heliothis virescens (13MacIntosh S.C. Stone T.B. Jokerst R.S. Fuchs R.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8930-8933Crossref PubMed Scopus (90) Google Scholar, 14Lee M.K. Rajamohan F. Gould F. Dean D.H. Appl. Environ. Microbiol. 1995; 61: 3836-3842Crossref PubMed Google Scholar), and beet armyworm, Spodoptera exigua (15Moar W.J. Pusztai-Carey M. van Faassen H. Bosch D. Frutos R. Rang C. Luo K. Adang M.J. Appl. Environ. Microbiol. 1995; 61: 2086-2092Crossref PubMed Google Scholar). However, reduced toxin binding is not always associated with resistance to Bt (16Gould F. Martinez-Ramirez A. Anderson A. Ferré J. Silva F.J. Moar W.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7986-7990Crossref PubMed Scopus (240) Google Scholar, 17Luo K. Tabashnik B.E. Adang M.J. Appl. Environ. Microbiol. 1997; 63: 1024-1027Crossref PubMed Google Scholar), and therefore alternate mechanisms of resistance must exist. A second mechanism of resistance may involve gut proteinases that interact with Bt toxins. Enzymes from a strain of H. virescens resistant to Bt subspecies kurstaki (HD-73) were reported to process the protoxin more slowly and to degrade toxin faster than enzymes from a susceptible strain (18Forcada C. Alcácer E. Garcerá M.D. Martı́nez R. Arch. Insect Biochem. Physiol. 1996; 31: 257-272Crossref Google Scholar). In Spodoptera littoralis, an increase in the specific activity of gut proteinases from fifth instar larvae was associated with a loss of sensitivity to Cry1C, possibly due to an increase in the degradation of toxin (19Keller M. Sneh B. Strizhov N. Prudovsky E. Regev A. Koncz C. Schell J. Zilberstein A. Insect Biochem. Mol. Biol. 1996; 26: 365-373Crossref PubMed Scopus (123) Google Scholar). Previously, we reported that a strain of P. interpunctellaresistant to Bt subspecies entomocidus has low soluble gut proteinase and Bt protoxin-hydrolyzing activities when compared with the parent-susceptible strain and a strain resistant to Bt subspecieskurstaki (20Oppert B.S. Kramer K.J. Johnson D.E. MacIntosh S.C. McGaughey W.H. Biochem. Biophys. Res. Commun. 1994; 198: 940-947Crossref PubMed Scopus (107) Google Scholar, 21Oppert B. Kramer K.J. Johnson D. Upton S.J. McGaughey W.H. Insect Biochem. Mol. Biol. 1996; 26: 571-583Crossref PubMed Scopus (87) Google Scholar). The slower protoxin hydrolysis observed with gut extracts from the entomocidus-resistant strain was an indication that proteinase-mediated mechanisms are involved in resistance to Bt. In this report, we characterize the proteolytic enzyme activity in Bt toxin-resistant insects and demonstrate a genetic linkage between the absence of a major gut proteinase and decreased susceptibility to the toxin. Late fourth instar larvae were chilled, and the posterior and anterior ends were removed. Guts were excised, immediately submersed in ice-cold 200 mm Tris, pH 8.0, 20 mm CaCl2 (buffer A), aliquoted 1 gut per 25 μl of buffer, and frozen at −20 °C for up to 2 weeks until assayed. For microplate proteinase assays, samples were thawed and spun at 15,000 × g for 2 min, and the supernatant containing soluble gut enzymes was used in assays. Samples were diluted 1:100 in buffer A, and 50 μl were added to a microplate well. N-α-Benzoyl-l-argininep-nitroanilide (BApNA, Sigma, 100 mg/ml in dimethyl sulfoxide) was diluted 1:100 in buffer A, and 50 μl were added to each well to initiate the reaction (final BApNA concentration was 1.15 mm). After a 30-s incubation at 37 °C, absorbance was monitored at 405 nm at 15-s intervals over a 5-min period. The change in absorbance per min was calculated by the software KinetiCalc3 (Biotek), and the data were converted to micromoles/min/mg of protein in each gut extract. Protein concentration was determined by the method of Bradford (22Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar) using bovine serum albumin as the standard in a microplate assay (Pierce). For proteinase activity blots, gut extracts from individual larvae or from five pooled individuals were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10–20% Tricine gels (Novex, 0.4 gut eq/lane). The proteinase activity blot procedure was performed as described previously (21Oppert B. Kramer K.J. Johnson D. Upton S.J. McGaughey W.H. Insect Biochem. Mol. Biol. 1996; 26: 571-583Crossref PubMed Scopus (87) Google Scholar). For zymogram analysis, gut samples were first analyzed for BApNA-hydrolyzing activity by microplate analysis. Volumes of samples were then adjusted for equivalent activity (ΔA 405 nm/min/ml = 2), and aliquots were subjected to SDS-PAGE on a 10–20% Tricine gel (Novex). Zymogram analysis was performed by incubating the gel with a Cry1Ac protoxin substrate solution (see below) for 2 h at 37 °C, followed by brief washes in deionized water and staining with Coomassie Blue, as described previously (21Oppert B. Kramer K.J. Johnson D. Upton S.J. McGaughey W.H. Insect Biochem. Mol. Biol. 1996; 26: 571-583Crossref PubMed Scopus (87) Google Scholar). Cry1Ac protoxin substrate solution was obtained from Bt subspecieskurstaki HD-73 grown on glucose/yeast extract/salts medium at 30 °C for 2–3 days with agitation until sporulation was complete (23Nickerson K.W. St. Julian G. Bulla L.A. Appl. Microbiol. 1974; 28: 129-132Crossref PubMed Google Scholar). Spores, crystals, and cellular debris were washed by centrifugation three times in phosphate-buffered saline and lyophilized. The lyophilized preparation was resuspended in 100 mm sodium carbonate, pH 10.0, 0.8 m sodium chloride, 10 mm EDTA, 20 mm dithiothreitol. After centrifuging at 1000 × g for 10 min, the supernatant was diluted 1:1 with deionized water and analyzed for Cry1Ac protoxin concentration by SDS-PAGE, Coomassie Blue staining, and scanning gel densitometry (International Technologies International) using homogenous Cry1Ac protoxin as the standard (data not shown). The concentration of Cry1Ac protoxin in the zymogram substrate solution was determined to be 0.3% and constituted greater than 50% of the total protein in the preparation. Fifteen virgin male adults from Bt-susceptible strain 688-s′, in which all individuals displayed the enzyme T1 (see “Results” for explanation of enzyme terminology), were mated with 15 virgin female entomocidus-resistant strain 198-r adults, which lacked T1. Gut extracts from approximately 15-day-old larvae from each strain were analyzed for BApNA hydrolytic patterns by proteinase activity blots. Fifteen female F1 adults were backcrossed to 15 resistant 198-r males, and progeny were analyzed for BApNA hydrolytic patterns. Female F1 were chosen because meiotic recombination does not occur in female Lepidoptera, and tight linkage would be observed between any pair of traits located on the same chromosome. Insects were reared on untreated diet throughout the matings. Ten mg of eggs from the backcrossed progeny were added to an untreated diet, and 15 mg were added to a 125 mg/kg Bt subspecies entomocidus(HD-198)-treated diet. Adults were selected from each group (n = 27 from the untreated group and n= 12 from the treated group) and placed on an untreated diet and allowed to mate. Progeny from each group were reared on an untreated diet, and guts were dissected from late fourth instar larvae. Gut extracts were analyzed for proteolytic patterns using proteinase activity blot analysis. Because trypsin-like proteinases in P. interpunctellaguts activate Bt protoxin (21Oppert B. Kramer K.J. Johnson D. Upton S.J. McGaughey W.H. Insect Biochem. Mol. Biol. 1996; 26: 571-583Crossref PubMed Scopus (87) Google Scholar), we examined trypsin-like enzyme activity in Bt-susceptible and resistant strains of the Indianmeal moth. Using the trypsin diagnostic substrate BApNA in a microplate assay, BApNA-hydrolyzing activities were detected in a Bt toxin-susceptible strain of P. interpunctella,688-s, and three resistant strains, 133-r, 198-r, and Dpl-r, derived from the susceptible parental strain via selection with spore/crystal preparations of Bt subspecies aizawai(HD-133), entomocidus (HD-198), or kurstaki(HD-1, Dipel®), 3Dipel® is a registered trademark of Abbott Laboratories. respectively (24McGaughey W.H. Johnson D.E. J. Econ. Entomol. 1992; 85: 1594-1600Crossref Google Scholar) (Table I). Specific activities of enzymes in the aizawai- andentomocidus-resistant strains were less than one-half of those in the kurstaki-resistant strain and in the parent-susceptible strain. Activity from thekurstaki-resistant strain was approximately 30% higher than activity from the parent-susceptible strain.Table IBApNA-hydrolyzing activity in gut extracts from a Bt-susceptible strain of P. interpunctella (688s) and strains resistant to Bt subspecies aizawai (133-r), entomocidus (198-r), and kurstaki (Dpl-r) maintained on treated dietsStrainSpecific activity1-aUnits are micromoles/min/mg of protein in gut extract (ε = 0.87 × 104m−1 cm−1, 0.3-cm path length). The number of individual insects used in each sample was 16 ± S.E.Activity relative to strain 688-s%688-s0.84 ± 0.09100133-r0.36 ± 0.0643198-r0.39 ± 0.0646Dpl-r1.06 ± 0.10126Extracts were analyzed for the ability to hydrolyze the substrateN-α-benzoyl-l-argininep-nitroanilide (BApNA).1-a Units are micromoles/min/mg of protein in gut extract (ε = 0.87 × 104m−1 cm−1, 0.3-cm path length). The number of individual insects used in each sample was 16 ± S.E. Open table in a new tab Extracts were analyzed for the ability to hydrolyze the substrateN-α-benzoyl-l-argininep-nitroanilide (BApNA). To determine the number and relative size of BApNA-hydrolyzing enzymes in each strain, larval gut extracts from the P. interpunctella strains were analyzed for their ability to hydrolyze BApNA using activity blots (Fig.1). Enzymes with relatively high molecular masses (>250 kDa) were observed in extracts from all four strains. Two major BApNA-hydrolyzing enzymes, designated T1 and T2 (apparent molecular masses ∼45 and ∼25 kDa, respectively), were observed in the Bt toxin-susceptible strain (688-s) and also in the Dipel-resistant strain (Dpl-r). The enzyme corresponding to T1, however, was not present in gut extracts from the strains resistant to either subspecies entomocidus (198-r) or subspeciesaizawai (133-r). A minor trypsin-like enzyme, T3, was observed in all of these strains, which had an apparent molecular mass of approximately 50 kDa. To determine whether these trypsin-like enzymes hydrolyze protoxin, an experiment was designed to compare the relative hydrolysis of protoxin by gut extracts from Bt-susceptible and -resistant P. interpunctella strains. Gut proteinases from the four P. interpunctella strains were separated electrophoretically by SDS-PAGE, and the gel was incubated in a solution of Bt subspecieskurstaki (HD-73) spores and crystals containing Cry1Ac protoxin as the major protein (Fig. 2). Dark zones in the zymogram demonstrated activity of enzymes from the gut extracts, which are capable of hydrolyzing proteins in the spore/crystal protein preparation. Major hydrolyzing activity corresponded to enzymes T1 and T2 in the susceptible andkurstaki-resistant strains but only T2 in theaizawai- and entomocidus-resistant strains. T1 and T2 have similar levels of activity. However, T2 appears to have a higher level of activity because it comigrates with another protoxin-hydrolyzing enzyme identified as one with chymotrypsin-like specificity (data not shown). No hydrolysis of protoxin by the higher molecular mass enzymes (i.e. the >250-kDa BApNA-hydrolyzing enzymes) was observed. Until now, BApNA-hydrolyzing enzymes had only been examined in extracts from pooled insects (Fig. 1). When gut extracts from individual susceptible larvae were used in enzyme activity blots, we observed that the strain consisted of a heterogeneous population of insects with respect to the expression of trypsin-like proteinases. Some insects expressed T1 and T2, whereas others expressed only T2 (data not shown). Therefore, single pair lines of susceptible strain 688-s were established to ensure genetic uniformity for subsequent linkage tests, and progeny were analyzed using proteinase activity blots. Progeny from one of these “isofemale” designated as 688-s′, were analyzed for four and it was determined that all individuals in this population expressed T1 and T2 trypsin-like enzymes blot is in To determine the T1 with toxin resistance interpunctella, analysis was Insects from the susceptible strain were mated with those from the entomocidus-resistant strain 198-r, and female F1 progeny were backcrossed to from the resistant strain. larvae from the parental the F1 and the were analyzed by proteinase activity blots for BApNA-hydrolyzing enzymes blots are in on the analysis of single gut extracts from each strain, the susceptible and resistant strains to be for the and of the T1 parental strains were for T2, all individuals displayed the T2 In the F1 individuals were examined and all T1 enzyme activity. However, the activity of T1 in the F1 larvae was than that in the susceptible strain, with the that the F1 were for the T1 the larvae analyzed for BApNA hydrolytic activity in the lacked the T1 enzyme, whereas expressed T1 and T2, which is to the 1:1 = of = = Insects from this population were then analyzed for the ability to on diets. eggs were placed on and mg of Bt subspecies entomocidus per of diet, and was A of 125 mg/kg was to all susceptible and resistant larvae (data not shown). at the three Bt toxin for the control were and respectively J. Econ. Entomol. Google Scholar). the 125 mg/kg of the insects the same of the that lacked the T1 enzyme when reared on untreated diet. The of the T1 enzyme in of progeny reared on untreated was determined by proteinase activity blots In the untreated 20 of 50 insects lacked the T1 trypsin-like whereas the had T1 and To in enzyme activity due to toxin adults from progeny on an untreated or diet mg/kg of were and placed on untreated diet to larvae were analyzed for proteinase patterns. In the progeny from untreated diet, 30 of 50 displayed T1 and T2 of the progeny from the on the Bt-treated diet of however, lacked the major trypsin-like proteinase T1. from a microplate assay of BApNA hydrolysis by P. interpunctella gut extracts demonstrated that activity was reduced in the aizawai- and entomocidus-resistant larvae when compared with kurstaki-resistant or susceptible larvae. have also observed slower protoxin hydrolysis with enzymes from strain, similar to results obtained with the entomocidus-resistant strain (20Oppert B.S. Kramer K.J. Johnson D.E. MacIntosh S.C. McGaughey W.H. Biochem. Biophys. Res. Commun. 1994; 198: 940-947Crossref PubMed Scopus (107) Google B. Kramer K.J. Johnson D. Upton S.J. McGaughey W.H. Insect Biochem. Mol. Biol. 1996; 26: 571-583Crossref PubMed Scopus (87) Google Scholar). K. J. and H. Therefore, resistance may be a result of reduced proteolytic activity in theaizawai- and entomocidus-resistant strains, but it is to proteinase activity in thekurstaki-resistant strain. The kurstaki-resistant strain is resistant due to a strain of P. S.S. Cowles E.A. Pietrantonio P.V. Annu. Rev. Entomol. 1992; 37: 615-636Crossref PubMed Google Scholar). from a proteinase activity blot assay using BApNA as the substrate that aizawai- andentomocidus-resistant larvae lack a major BApNA-hydrolyzing enzyme, T1. The absence of T1 activity in these strains was also in zymogram analysis of Bt activity. T1 is a protoxin-hydrolyzing enzyme, the lack of an T1 in theaizawai- and entomocidus-resistant strains would result in less toxin in the that the absence of this T1 enzyme to reduced of protoxin a survival when insects on containing Bt toxins. of susceptible insects that have T1 and T2 enzymes with resistant insects that have only the T1 enzyme in progeny with T2 activity but only a relatively low T1 activity. A of these insects to the resistant strain in progeny of a of with individuals either T1 and T2 or only larvae that a of Bt toxin lacked the T1 Therefore, when insects not have an T1, are to a of toxin that is to insects that are or for T1. These data demonstrate a genetic linkage between resistance and the absence of T1 and are evidence for a proteinase-mediated mechanism of resistance to Bt. of genes that have by electrophoresis are and are N. G. F. J. J. Scholar). In the absence of the enzyme is due to a that results in an protein or in are the described. The number of of a for in is more than times greater in than in strains N. de B. de M. Science. PubMed Scopus Google Scholar). In that the is for insects. In P. interpunctella, however, the is a survival for the insects. is the first of a for a trypsin-like proteinase of which we are In we have demonstrated that Bt toxin-resistant strains of P. interpunctella have BApNA-hydrolyzing and than a susceptible or another resistant strain. These are due to the lack of a major gut trypsin-like proteinase in the resistant strains. of a genetic analysis demonstrated that insect resistance to Bt toxins with the loss of this major trypsin-like The absence of the trypsin-like enzyme results in reduced levels of toxin in the the insects to to high levels of toxin. has to the understanding that the genetic in the insect population and the of the toxin preparation resistance for reduced proteinase activity is with only Bt However, these preparations will not for a the is not present in the K. J. and H. is not Bt that different resistance in insects. the for resistance in insect to different Bt will provide for more effective toxin and in pest management that Bt toxins. the by and

Switchgrass ( Panicum virgatum L.) is a widely adapted warm‐season perennial that has considerable potential as a biofuel crop. Evolutionary processes and environmental factors have combined to create considerable ecotypic differentiation in switchgrass. The objective of this study was to determine the nature of population × location interaction for switchgrass, quantifying potential differences in latitudinal adaptation of switchgrass populations. Twenty populations were evaluated for biofuel and agronomic traits for 2 yr at five locations ranging from 36 to 46° N lat. Biomass yield, survival, and plant height had considerable population × location interaction, much of which (53–65%) could be attributed to the linear effect of latitude and to germplasm groups (Northern Upland, Southern Upland, Northern Lowland, and Southern Lowland). Differences among populations were consistent across locations for maturity, dry matter, and lodging. Increasingly later maturity and the more rapid stem elongation rate of more southern‐origin ecotypes (mainly lowland cytotypes) resulted in high biomass yield potential, reduced dry matter concentration, and longer retention of photosynthetically active tissue at more southern locations. Conversely, increasing cold tolerance of more northern‐origin ecotypes (mainly upland cytotypes) resulted in higher survival, stand longevity, and sustained biomass yields at more northern locations, allowing switchgrass to thrive at cold, northern latitudes. Although cytotype explained much of the variation among populations and the population × location interaction, ecotypic differentiation within cytotypes accounted for considerable variation in adaption of switchgrass populations.

BACKGROUND: Relatively little is known about the genomic basis and evolution of wood-feeding in beetles. We undertook genome sequencing and annotation, gene expression assays, studies of plant cell wall degrading enzymes, and other functional and comparative studies of the Asian longhorned beetle, Anoplophora glabripennis, a globally significant invasive species capable of inflicting severe feeding damage on many important tree species. Complementary studies of genes encoding enzymes involved in digestion of woody plant tissues or detoxification of plant allelochemicals were undertaken with the genomes of 14 additional insects, including the newly sequenced emerald ash borer and bull-headed dung beetle. RESULTS: The Asian longhorned beetle genome encodes a uniquely diverse arsenal of enzymes that can degrade the main polysaccharide networks in plant cell walls, detoxify plant allelochemicals, and otherwise facilitate feeding on woody plants. It has the metabolic plasticity needed to feed on diverse plant species, contributing to its highly invasive nature. Large expansions of chemosensory genes involved in the reception of pheromones and plant kairomones are consistent with the complexity of chemical cues it uses to find host plants and mates. CONCLUSIONS: Amplification and functional divergence of genes associated with specialized feeding on plants, including genes originally obtained via horizontal gene transfer from fungi and bacteria, contributed to the addition, expansion, and enhancement of the metabolic repertoire of the Asian longhorned beetle, certain other phytophagous beetles, and to a lesser degree, other phytophagous insects. Our results thus begin to establish a genomic basis for the evolutionary success of beetles on plants.

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19) and responsible for the current pandemic. Recent SARS-CoV-2 susceptibility studies in cats show that the virus can replicate in these companion animals and transmit to other cats. Here, we present an in-depth study of SARS-CoV-2 infection, disease and transmission in domestic cats. Cats were challenged with SARS-CoV-2 via intranasal and oral routes. One day post challenge (DPC), two sentinel cats were introduced. Animals were monitored for clinical signs, clinicopathological abnormalities and viral shedding. Postmortem examinations were performed at 4, 7 and 21 DPC. Viral RNA was not detected in blood but transiently in nasal, oropharyngeal and rectal swabs and bronchoalveolar lavage fluid as well as various tissues. Tracheobronchoadenitis of submucosal glands with the presence of viral RNA and antigen was observed in airways of the infected cats. Serology showed that both, principals and sentinels, developed antibodies to SARS-CoV-2. All animals were clinically asymptomatic during the course of the study and capable of transmitting SARS-CoV-2 to sentinels. The results of this study are critical for understanding the clinical course of SARS-CoV-2 in a naturally susceptible host species, and for risk assessment.

Phosphorous in soybean [ Glycine max (L.) Merr.] seed is stored primarily as phytic acid, which is nutritionally unavailable to nonruminant livestock. The objective of this study was to isolate mutations that reduce soybean seed phytic acid P and increase seed inorganic P. Following treatment with ethyl methanesulfonate, M2 through M6 plants were screened for high seed inorganic P. Seeds of M2 plants high in inorganic P produced progenies high in inorganic P through the M6 generation. M6 progenies of one plant averaged 6.84 g kg −1 seed phytic acid and inorganic P varied from 2.34 to 4.41 g kg −1 or 60 to 66% of phytic acid P plus inorganic P. M6 progenies of a second plant averaged 10.89 g kg −1 phytic acid and varied from 1.21 to 3.84 g kg −1 inorganic P, representing from 47 to 51% of the sum of phytic acid P plus inorganic P. In contrast, nonmutant seeds of the check cultivar Athow contained 15.33 g kg −1 phytic acid and averaged 0.74 g kg −1 inorganic P, representing 15% of the sum of phytic acid P plus inorganic P. Low phytic acid and high inorganic P in these progenies should increase the nutritional value of soy meal and reduce excess P in livestock manure.