Resistance Bioassays and Allele Characterization Inform Analysis of Spodoptera frugiperda (Lepidoptera: Noctuidae) Introduction Pathways in Asia and Australia.

The fall armyworm (FAW) Spodoptera frugiperda (Smith; Lepidoptera: Noctuidae) is present in over 70 countries in Africa, Asia, and Oceania. Its rapid dispersal since 2016 when it was first reported in western Africa, and associated devastation to agricultural productivity, highlight the challenges posed by this pest. Currently, its management largely relies on insecticide sprays and transgenic Bacillus thuringiensis toxins, therefore understanding their responses to these agents and characteristics of any resistance genes enables adaptive strategies. In Australia, S. frugiperda was reported at the end of January 2020 in northern Queensland and by March 2020, also in northern Western Australia. As an urgent first response we undertook bioassays on two Australian populations, one each from these initial points of establishment. To assist with preliminary sensitivity assessment, two endemic noctuid pest species, Helicoverpa armigera (Hübner; Lepidoptera, Noctuidae) and Spodoptera litura (Fabricius; Lepidoptera, Noctuidae), were concurrently screened to obtain larval LC50 estimates against various insecticides. We characterized known resistance alleles from the VGSC, ACE-1, RyR, and ABCC2 genes to compare with published allele frequencies and bioassay responses from native and invasive S. frugiperda populations. An approximately 10× LC50 difference for indoxacarb was detected between Australian populations, which was approximately 28× higher than that reported from an Indian population. Characterization of ACE-1 and VGSC alleles provided further evidence of multiple introductions in Asia, and multiple pathways involving genetically distinct individuals in Australia. The preliminary bioassay results and resistance allele patterns from invasive S. frugiperda populations suggest multiple introductions have contributed to the pest's spread and challenge the axiom of its rapid 'west-to-east' spread.

Determinants of Insecticide Resistance Evolution: Comparative Analysis Among Heliothines.

It is increasingly clear that pest species vary widely in their propensities to develop insecticide resistance. This review uses a comparative approach to analyze the key pest management practices and ecological and biochemical or genetic characteristics of the target that contribute to this variation. We focus on six heliothine species, three of which, Helicoverpa armigera, Heliothis virescens, and Helicoverpa zea, have developed resistances to many pesticide classes. The three others, Helicoverpa punctigera, Helicoverpa assulta, and Helicoverpa gelotopoeon, also significant pests, have developed resistance to very few pesticide classes. We find that host range and movement between alternate hosts are key ecological traits that influence effective selection intensities for resistance. Operational issues are also critical; area-wide, cross-pesticide management practices that account for these ecological factors are key to reducing selection intensity. Without such management, treatment using broad-spectrum chemicals serves to multiply the effects of host plant preference, preadaptive detoxification ability, and high genetic diversity to create a pesticide treadmill for the three high-propensity species.Without rigorous ongoing management, such a treadmill could still develop for newer, more selective chemistries and insecticidal transgenic crops.

On species delimitation, hybridization and population structure of cassava whitefly in Africa.

The Bemisia cassava whitefly complex includes species that cause severe crop damage through vectoring cassava viruses in eastern Africa. Currently, this whitefly complex is divided into species and subgroups (SG) based on very limited molecular markers that do not allow clear definition of species and population structure. Based on 14,358 genome-wide SNPs from 62 Bemisia cassava whitefly individuals belonging to sub-Saharan African species (SSA1, SSA2 and SSA4), and using a well-curated mtCOI gene database, we show clear incongruities in previous taxonomic approaches underpinned by effects from pseudogenes. We show that the SSA4 species is nested within SSA2, and that populations of the SSA1 species comprise well-defined south-eastern (Madagascar, Tanzania) and north-western (Nigeria, Democratic Republic of Congo, Burundi) putative sub-species. Signatures of allopatric incipient speciation, and the presence of a 'hybrid zone' separating the two putative sub-species were also detected. These findings provide insights into the evolution and molecular ecology of a highly cryptic hemipteran insect complex in African, and allow the systematic use of genomic data to be incorporated in the development of management strategies for this cassava pest.

Genomic innovations, transcriptional plasticity and gene loss underlying the evolution and divergence of two highly polyphagous and invasive Helicoverpa pest species.

BACKGROUND: Helicoverpa armigera and Helicoverpa zea are major caterpillar pests of Old and New World agriculture, respectively. Both, particularly H. armigera, are extremely polyphagous, and H. armigera has developed resistance to many insecticides. Here we use comparative genomics, transcriptomics and resequencing to elucidate the genetic basis for their properties as pests. RESULTS: We find that, prior to their divergence about 1.5 Mya, the H. armigera/H. zea lineage had accumulated up to more than 100 more members of specific detoxification and digestion gene families and more than 100 extra gustatory receptor genes, compared to other lepidopterans with narrower host ranges. The two genomes remain very similar in gene content and order, but H. armigera is more polymorphic overall, and H. zea has lost several detoxification genes, as well as about 50 gustatory receptor genes. It also lacks certain genes and alleles conferring insecticide resistance found in H. armigera. Non-synonymous sites in the expanded gene families above are rapidly diverging, both between paralogues and between orthologues in the two species. Whole genome transcriptomic analyses of H. armigera larvae show widely divergent responses to different host plants, including responses among many of the duplicated detoxification and digestion genes. CONCLUSIONS: The extreme polyphagy of the two heliothines is associated with extensive amplification and neofunctionalisation of genes involved in host finding and use, coupled with versatile transcriptional responses on different hosts. H. armigera's invasion of the Americas in recent years means that hybridisation could generate populations that are both locally adapted and insecticide resistant.

Population structure and gene flow in the global pest, Helicoverpa armigera.

Helicoverpa armigera is a major agricultural pest that is distributed across Europe, Asia, Africa and Australasia. This species is hypothesized to have spread to the Americas 1.5 million years ago, founding a population that is at present, a distinct species, Helicoverpa zea. In 2013, H. armigera was confirmed to have re-entered South America via Brazil and subsequently spread. The source of the recent incursion is unknown and population structure in H. armigera is poorly resolved, but a basic understanding would highlight potential biosecurity failures and determine the recent evolutionary history of region-specific lineages. Here, we integrate several end points derived from high-throughput sequencing to assess gene flow in H. armigera and H. zea from populations across six continents. We first assemble mitochondrial genomes to demonstrate the phylogenetic relationship of H. armigera with other Heliothine species and the lack of distinction between populations. We subsequently use de novo genotyping-by-sequencing and whole-genome sequences aligned to bacterial artificial chromosomes, to assess levels of admixture. Primarily, we find that Brazilian H. armigera are derived from diverse source populations, with strong signals of gene flow from European populations, as well as prevalent signals of Asian and African ancestry. We also demonstrate a potential field-caught hybrid between H. armigera and H. zea, and are able to provide genomic support for the presence of the H. armigera conferta subspecies in Australasia. While structure among the bulk of populations remains unresolved, we present distinctions that are pertinent to future investigations as well as to the biosecurity threat posed by H. armigera.

Soybean Stem Fly, Melanagromyza sojae (Diptera: Agromyzidae), in the New World: detection of high genetic diversity from soybean fields in Brazil.

Soybean Stem Fly (SSF), Melanagromyza sojae (Zehntner), belongs to the family Agromyzidae and is highly polyphagous, attacking many plant species of the family Fabaceae, including soybean and other beans. SSF is regarded as one of the most important pests in soybean fields of Asia (e.g., China, India), North East Africa (e.g., Egypt), parts of Russia, and South East Asia. Despite reports of Agromyzidae flies infesting soybean fields in Rio Grande do Sul State (Brazil) in 1983 and 2009 and periodic interceptions of SSF since the 1940s by the USA quarantine authorities, SSF has not been officially reported to have successfully established in the North and South Americas. In South America, M. sojae was recently confirmed using morphology and its complete mitochondrial DNA (mtDNA) was characterized. In the present study, we surveyed the genetic diversity of M. sojae, collected directly from soybean host plants, using partial mtDNA cytochrome oxidase I (COI) gene, and provide evidence of multiple (>10) maternal lineages in SSF populations in South America, potentially representing multiple incursion events. However, a single incursion involving multiple-female founders could not be ruled out. We identified a haplotype that was common in the fields of two Brazilian states and the individuals collected from Australia in 2013. The implications of SSF incursions in southern Brazil are discussed in relation to the current soybean agricultural practices, highlighting an urgent need for better understanding of SSF population movements in the New World, which is necessary for developing effective management options for this significant soybean pest.

Mitochondrial DNA COI characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) from Paraguay and Uruguay.

Since its detection in Brazil in 2013, the Old World cotton bollworm Helicoverpa armigera has been reported in Argentina, Paraguay, and Bolivia. Here we present evidence extending the South American range of H. armigera to Uruguay, using polymerase chain reaction and sequencing of the partial mitochondrial DNA (mtDNA) cytochrome oxidase I region. Molecular characterization of this gene region from individuals from Paraguay also supports previous morphological identification of H. armigera in Paraguay. Shared mtDNA haplotypes in H. armigera from Brazil, Uruguay, and Paraguay were identified. Additional surveying of populations in this region will be imperative to better monitor and understand factors that are underpinning its presence and successful adaptation in these South American regions. We discuss our findings with respect to the development of resistance pest management strategies of this invasive insect pest in a predominantly monoculture soybean crop landscape in the Southern Cone region.

Dual Cry2Ab and Vip3A resistant strains of Helicoverpa armigera and Helicoverpa punctigera (Lepidoptera: Noctuidae); testing linkage between loci and monitoring of allele frequencies.

Considerable attention has been given to delaying the evolution of insect resistance to toxins produced by transgenic crops. The major pests of cotton in Australia are the Lepidoptera Helicoverpa armigera (Hubner, 1805) and Helicoverpa punctigera (Wallengren), and the toxins deployed in current and imminent transgenic cotton varieties are Cry1Ac, Cry2Ab and Vip3A from Bacillus thuringiensis. In this study, lines that carry alleles conferring resistance to Cry2Ab and Vip3A were isolated using F2 tests. Extensive work on the Cry2Ab resistant lines, and preliminary work on the Vip3A resistant lines, suggested a single common resistance to each toxin in both species thereby justifying the use of more efficient F1 tests as the primary means for monitoring changes over time. A potential further efficiency could be gained by developing a single resistant line that carries both types of Bt resistance. Herein we report on work with both H. armigera and H. punctigera that tests whether dual Cry2Ab-Vip3A resistant lines can be developed and, if so, whether they can be used to effectively monitor resistance frequencies. Furthermore, the creation of dual resistant lines allowed linkage between the Cry2Ab and Vip3A resistances to be investigated for H. punctigera. We show that dual resistant lines can be used to increase the efficiency of the F1 screen for recessive alleles, and that in H. punctigera there is no linkage between Cry2Ab and Vip3A resistance.

Juvenile hormone titre and related gene expression during the change of reproductive modes in the pea aphid.

Most aphids show reproductive polyphenism, i.e. they alternate their reproductive modes from parthenogenesis to sexual reproduction in response to short photoperiods. Although juvenile hormone (JH) has been considered a likely candidate for regulating the transition from asexual to sexual reproduction after photoperiod sensing, there are few studies investigating the direct relationship between JH titres and the reproductive-mode change. In addition, the sequencing of the pea aphid genome has allowed identification of the genes involved in the JH pathway, which in turn allows us to examine their expression levels in relation to the reproductive-mode change. Using liquid chromatography-mass spectrometry in the pea aphid, JHIII titre was shown to be lower in aphids producing sexual morphs under short-day conditions than in aphids producing parthenogenetic morphs under long-day conditions. The expression levels of genes upstream and downstream of JH action were quantified by real-time quantitative reverse-transcription-PCR across the reproductive-mode change. The expression level of JH esterase, which is responsible for JH degradation, was significantly higher in aphids reared under short-day conditions. This suggests that the upregulation of the JH degradation pathway may be responsible for the lower JHIII titre in aphids exposed to short-days, leading to the production of sexual morphs.

Identification of ion channel genes in the Acyrthosiphon pisum genome.

Aphids are major pests of crops, causing hundreds of millions of dollars worth of damage annually. Ion channel proteins are often the targets of modern insecticides and mutations in ion channel genes can lead to resistance to many leading classes of insecticides. The sequencing of the pea aphid, Acyrthosiphon pisum, genome has now allowed detailed in silico analysis of the aphid ion channels. The study has revealed significant differences in the composition of the ion channel families between the aphid and other insects. For example A. pisum does not appear to contain a homologue of the nACh receptor alpha 5 gene whilst the calcium channel beta subunit has been duplicated. These variations could result in differences in function or sensitivity to insecticides. The genome sequence will allow the study of aphid ion channels to be accelerated, leading to a better understanding of the function of these economically important channels. The potential for identifying novel insecticide targets within the aphid is now a step closer.

Comparative analysis of detoxification enzymes in Acyrthosiphon pisum and Myzus persicae.

Herbivorous insects use detoxification enzymes, including cytochrome P450 monooxygenases, glutathione S-transferases, and carboxy/cholinesterases, to metabolize otherwise deleterious plant secondary metabolites. Whereas Acyrthosiphon pisum (pea aphid) feeds almost exclusively from the Fabaceae, Myzus persicae (green peach aphid) feeds from hundreds of species in more than forty plant families. Therefore, M. persicae as a species would be exposed to a greater diversity of plant secondary metabolites than A. pisum, and has been predicted to require a larger complement of detoxification enzymes. A comparison of M. persicae cDNA and A. pisum genomic sequences is partially consistent with this hypothesis. There is evidence of at least 40% more cytochrome P450 genes in M. persicae than in A. pisum. In contrast, no major differences were found between the two species in the numbers of glutathione S-transferases, and carboxy/cholinesterases. However, given the incomplete M. persicae cDNA data set, the number of identified detoxification genes in this species is likely to be an underestimate.

A functional DNA methylation system in the pea aphid, Acyrthosiphon pisum.

Methylation of cytosine is one of the main epigenetic mechanisms involved in controlling gene expression. Here we show that the pea aphid (Acyrthosiphon pisum) genome possesses homologues to all the DNA methyltransferases found in vertebrates, and that 0.69% (+/-0.25%) of all cytosines are methylated. Identified methylation sites are predominantly restricted to the coding sequence of genes at CpG sites. We identify twelve methylated genes, including genes that interact with juvenile hormone, a key endocrine signal in insects. Bioinformatic prediction using CpG ratios for all predicted genes suggest that a large proportion of genes are methylated within the pea aphid.

Molecular detection of benzimidazole resistance in Haemonchus contortus using real-time PCR and pyrosequencing.

Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in beta-tubulin genes have been associated with BZ-resistance. In the present study, we investigated beta-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000-10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the 'egg hatch test', and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.

Nematode ligand-gated chloride channels: an appraisal of their involvement in macrocyclic lactone resistance and prospects for developing molecular markers.

SUMMARYLigand-gated chloride channels, including the glutamate-(GluCl) and GABA-gated channels, are the targets of the macrocyclic lactone (ML) family of anthelmintics. Changes in the sequence and expression of these channels can cause resistance to the ML in laboratory models, such as Caenorhabditis elegans and Drosophila melanogaster. Mutations in multiple GluCl subunit genes are required for high-level ML resistance in C. elegans, and this can be influenced by additional mutations in gap junction and amphid genes. Parasitic nematodes have a different complement of channel subunit genes from C. elegans, but a few genes, including avr-14, are widely present. A polymorphism in an avr-14 orthologue, which makes the subunit less sensitive to ivermectin and glutamate, has been identified in Cooperia oncophora, and polymorphisms in several subunits have been reported from resistant isolates of Haemonchus contortus. This has led to suggestions that ML resistance may be polygenic. Possible reasons for this, and its consequences for the development of molecular tests for resistance, are explored.

In-vivo tissue uptake and retention of Sn-117m(4+)DTPA in a human subject with metastatic bone pain and in normal mice.

Organ and tissue uptake and retention of Sn-117m(4+)DTPA were studied in a human subject treated for metastatic bone pain, and the results were compared with the biodistribution studies in five normal mice. The explanted organs from a patient who received a therapy dose of 18.6 mCi (688.2 MBq) Sn-117m(4+)DTPA and who died 47 days later were imaged with a gamma-camera, and tissue samples were counted and also autoradiographed. Bone, muscle, liver, fat, lungs, kidneys, spleen, heart and pancreas tissue samples were assayed in a well counter for radioactivity. Regions of interest were drawn over bone and major organs to calculate and quantify clearance times using three in vivo Sn-117m(4+)DTPA whole-body scintigrams acquired at 1, 24 and 168 h after injection. Five normal mice injected with the same batch of Sn-117m(4+)DTPA as used for the human subject were sacrificed at 24 h, and tissue samples were collected and assayed for radioactivity for comparison with the human data. For the human subject, whole-body retention at 47 days postinjection was 81% of the injected dose, and the rest (19%) was excreted in urine. Of the whole-body retained activity at 47 days, 82.4% was in bone, 7.8% in the muscle and 1.5% in the liver, and the rest was distributed among other tissues. Gamma-ray scintigrams and electron autoradiographs of coronal slices of the thoracolumbar vertebral body showed heterogeneous metastatic involvement with normal bone between metastatic lesions. There was nonuniform distribution of radioactivity even within a single vertebral body, indicating normal bone between metastatic lesions. Lesion-to-nonlesion ratios ranged from 3 to 5. However, the osteoid-to-marrow cavity deposition ratio, from the microautoradiographs, was 11:1. The peak uptake in the human bone was seen at 137 h with no biological clearance. Soft tissues showed peak uptake at 1 h and exhibited three compartmental clearance components. Whole-body retention in normal mice was 38.7% of the injected dose at 24 h and the rest was excreted. At 24 h postinjection, bone in mice showed 84.2% of the whole-body retention, muscle 1.7% and liver 1.4%, and the rest was distributed in other soft tissues. Percent distribution of the retained dose among bone, muscle, liver and other soft tissues is very similar between mice and a human subject. To calculate precise radiation absorbed doses from bone pain palliation radionuclides, it is necessary to take into account soft-tissue uptake and retention that may not be readily evident from routine external gamma-scintigraphy.

Tin-117m(4+)DTPA: pharmacokinetics and imaging characteristics in patients with metastatic bone pain.

UNLABELLED: Biokinetics and imaging characteristics of 117mSn(4+)DTPA have been studied in patients with metastatic bone pain. METHODS: Seventeen patients with bone pain due to metastasis were given three dose levels: 180 microCi/kg (6.66 MBq/kg), 229 microCi/kg (8.47 MBq/kg) and 285 microCi/kg (10.55 MBq/kg) body weight. Periodic blood and daily urine samples were collected for 14 days to measure percent injected activity retained in blood and that excreted in urine. Simultaneous anterior and posterior view whole-body images were obtained under identical scan settings at 1, 3.5 and 24 hr and on Days 3 and 7 and between 4-6 and 8-10 wk postinjection. The total body retention was calculated using the geometric mean counts. RESULTS: After intravenous injection, the total body clearance of 117mSn(4+)DTPA shows two components: a soft-tissue component and a bone component. The soft-tissue component accounts for 22.4% of the dose and consists of four subcomponents with an average biologic clearance half-time of 1.45 days (range 0.1-3.2 days). The bone component accounting for the remaining 77.6% of the dose shows no biologic clearance. A mean 22.4% of the dose is excreted in urine in 14 days; 11.4% within 24 hr. The uptake pattern appears similar to that of 99mTc-MDP. Peak uptake is observed in normal bone by 24 hr and metastatic lesions by 3-7 days. Pain palliation was observed with all three doses levels. CONCLUSION: Among the four potential bone pain palliation radionuclides, 117mSn(4+)DTPA demonstrates the highest bone uptake and retention. Some biokinetic and radionuclidic features of 117mSn(4+)DTPA are similar to other agents, but many features are different and unique and may make it an ideal bone pain palliation agent. Double-blind comparative studies are needed to determine its exact role in bone pain palliation.

Ethanol and heart disease. An underestimated contributing factor.

With the number of chronic heavy users of ethanol in the United States estimated to be 15 to 20 million and the evidence increasing that ethanol causes serious cardiac metabolic disturbances, ethanol abuse is obviously a serious problem and most likely is an important contributing factor to cardiac morbidity and mortality. However, a direct cause-and-effect relationship between the biochemical dysfunctions produced by ethanol and the clinical entity of alcoholic cardiomyopathy has not been clearly established. What is lacking is a method to differentiate the damage secondary to ethanol abuse from that secondary to other causes. Sorely needed is a biochemical or anatomic marker (perhaps evaluated by serial myocardial biopsy) for alcoholic cardiomyopathy and a study to detect which cases of dilated cardiomyopathy indeed are due to ethanol-induced damage. Further longterm studies are also needed to demonstrate the benefits of abstinence upon large groups of patients, the effects of abstinence upon sudden death, and the effects of discontinuance of ethanol use for patients in the early stages of alcoholic cardiomyopathy. Ethanol is probably an underestimated contributing factor to cardiac disease. The importance of determining ethanol's impact on cardiovascular morbidity and mortality is underscored by the facts that alcoholic heart disease is completely avoidable and is largely reversible by abstinence.

Sarcoidosis mimicking cor triatriatum. Echolucency of adenopathy due to sarcoidosis.

A 20-year-old man with sarcoidosis presented with the echocardiographic findings of cor triatriatum. Computed tomography of the chest and digital subtraction angiography of the heart revealed that the patient had massive retrocardiac lymphadenopathy and normal cardiac anatomy. This is the first report demonstrating the echolucency of sarcoid lymphadenopathy and the mimicking of cor triatriatum by such adenopathy fortuitously positioned.