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PDF file - 110K, Non-synonymous somatic mutations identified in the Discovery set of four NKTCL cases

PDF file - 83K, JAK1 and JAK3 mutation status in NKTCL cases

PDF file - 75K, Sequence analysis summary of tumor and blood samples from four NKTCL cases

PDF file - 74K, Primer sets used for Sanger sequencing and HRM analysis

PDF file - 185K, Representative HRM curves of a NKTCL case confirmed as heterozygous JAK3A573V mutation

The Nobel Prize-winning discovery that human somatic cells can be readily reprogrammed into pluripotent cells has revolutionized our potential to understand the human brain. The rapid technological progression of this field has made it possible to easily obtain human neural cells and even intact tissues, offering invaluable resources to model human brain development. In this chapter, we present a brief history of hPSC-based approaches to study brain development and then, provide new insights into neurological diseases, focusing on those driven by aberrant cell death. Furthermore, we will shed light on the latest technologies and highlight the methods that researchers can use to employ established hPSC approaches in their research. Our intention is to demonstrate that hPSC-based modeling is a technical approach accessible to all researchers who seek a deeper understanding of the human brain.

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m(5)dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N(6)-methyldeoxyadenosine (m(6)dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m(6)dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.

Background Regeneration of healthy lung tissue in patients with end-stage respiratory disease would cure disease. Therefore, a detailed understanding of lung development is needed. The most important epithelial stem-cell population in developing lungs is found in distal branching tips, and these Sox9+ lung epithelial stem cells (LESCs) generate all epithelial lineages. Our objective was to develop a self-renewing, genetically modifiable epithelial in-vitro culture system from human embryonic LESCs. Methods Human embryonic LESCs were characterised using genome-wide transcriptional analysis (RNA-seq, Illumina, San Diego, CA, USA) and immunohistochemistry at 5–20 weeks postconception. LESCs were microdissected, and self-renewing expansion in three-dimensional (3D) organoid culture was established empirically. We assessed similarity between cultured and fresh LESCs via RNA-seq and immunohistochemistry. Organoids were differentiated in vitro, or in vivo by xenotransplantation into bleomycin-injured mouse lungs or kidney capsule. SOX9 was deleted with CRISPR–Cas9. Findings RNA-seq of LESCs identified broad-scale transcriptional differences between mouse and human embryonic lung stem cells. Human LESCs were successfully expanded for more than 10 months as karyotypically stable 3D organoids using a combination of seven signalling molecules. The LESC stem-cell markers, transcriptome, and organoid morphology were maintained throughout the culture period. Bronchiolar and alveolar differentiation was achieved in vitro and in vivo. Moreover, xenotransplantation of organoids into bleomycin-injured adult mouse lungs was extremely efficient. Knocking out SOX9 led to a loss-of-self-renewing phenotype. Interpretation Our novel genetically modifiable human embryonic lung culture system enables for the first time, to our knowledge, the in-vitro study of human lung development and disease. We anticipate that this system will transform lung regenerative medicine by guiding development of improved protocols for induced pluripotent stem-cell differentiation and adult stem-cell manipulation in vivo, benefiting patients with end-stage respiratory disease. Furthermore, our lung injury–xenotransplantation protocol provides the first method for efficient future cell therapy. Funding Wellcome Trust (PhD programme for clinicians), Medical Research Council, Addenbrooke's Charitable Trust.

Summary Oocytes have a remarkable ability to reactivate silenced genes in somatic cells. However, it is not clear how the chromatin architecture of somatic cells affects this transcriptional reprogramming. Here, we investigated the relationship between the chromatin opening and transcriptional activation. We reveal changes in chromatin accessibility and their relevance to transcriptional reprogramming after transplantation of somatic nuclei into Xenopus oocytes. Genes that are silenced, but have pre-existing open transcription start sites in donor cells, are prone to be activated after nuclear transfer, suggesting that the chromatin signature of somatic nuclei influences transcriptional reprogramming. There are also activated genes associated with new open chromatin sites, and transcription factors in oocytes play an important role in transcriptional reprogramming from such genes. Finally, we show that genes resistant to reprogramming are associated with closed chromatin configurations. We conclude that chromatin accessibility is a central factor for successful transcriptional reprogramming in oocytes., Graphical Abstract, Highlights • ATAC-seq reveals chromatin accessibility changes during reprogramming in oocytes • Genes with open promoters are preferentially activated during reprogramming • Transcription factors play a role in transcriptional reprogramming in oocytes • Closed chromatin is associated with reprogramming-resistant genes, Miyamoto et al. show genome-wide changes in chromatin accessibility during transcriptional reprogramming in oocytes using the frog nuclear transfer system. They demonstrate that donor cell chromatin states affect transcriptional reprogramming and changes in open chromatin during reprogramming are associated with specific transcription factors.

dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses.

dA6m DNA immunoprecipitation followed by deep sequencing (DIP-Seq) is a key tool in identifying and studying the genome-wide distribution of N6-methyldeoxyadenosine (dA6m). The precise function of this novel DNA modification remains to be fully elucidated, but it is known to be absent from transcriptional start sites and excluded from exons, suggesting a role in transcriptional regulation (Koziol et al., 2015). Importantly, its existence suggests that DNA might be more diverse than previously believed, as further DNA modifications might exist in eukaryotic DNA (Koziol et al., 2015). This protocol describes the method to perform dA6m DNA immunoprecipitation (DIP), as was applied to characterize the first dA6m methylome analysis in higher eukaryotes (Koziol et al., 2015). In this protocol, we describe how genomic DNA is isolated, fragmented and then DNA containing dA6m is pulled down with an antibody that recognizes dA6m in genomic DNA. After subsequent washes, DNA fragments that do not contain dA6m are eliminated, and the dA6m containing fragments are eluted from the antibody in order to be processed further for subsequent analyses. BACKGROUND: This protocol was developed in order to identify regions in the genome that contain dA6m. It can be used to detect dA6m in different genomes. As a guideline, this protocol was established from existing approaches used to detect adenosine methylation in RNA (Dominissini et al., 2013). We developed this protocol and adapted it for the detection of dA6m in DNA, rather than detecting adenosine methylation RNA. This was required, as no protocol was available at that time to allow the genome-wide identification of dA6m in eukaryotic DNA.

The molecular pathogenesis of natural killer/T-cell lymphoma (NKTCL) is not well understood. We conducted whole-exome sequencing and identified Janus kinase 3 (JAK3) somatic–activating mutations (A572V and A573V) in 2 of 4 patients with NKTCLs. Further validation of the prevalence of JAK3 mutations was determined by Sanger sequencing and high-resolution melt (HRM) analysis in an additional 61 cases. In total, 23 of 65 (35.4%) cases harbored JAK3 mutations. Functional characterization of the JAK3 mutations support its involvement in cytokine-independent JAK/STAT constitutive activation leading to increased cell growth. Moreover, treatment of both JAK3-mutant and wild-type NKTCL cell lines with a novel pan-JAK inhibitor, CP-690550, resulted in dose-dependent reduction of phosphorylated STAT5, reduced cell viability, and increased apoptosis. Hence, targeting the deregulated JAK/STAT pathway could be a promising therapy for patients with NKTCLs. Significance: Gene mutations causing NKTCL have not been fully identified. Through exome sequencing, we identified activating mutations of JAK3 that may play a significant role in the pathogenesis of NKTCLs. Our findings have important implications for the management of patients with NKTCLs. Cancer Discov; 2(7); 591–7. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 569.

Bin Tean Teh and colleagues report exome sequencing of Opisthorchis viverrini–related cholangiocarcinoma, a fatal bile duct cancer associated with liver fluke infection. Opisthorchis viverrini–related cholangiocarcinoma (CCA), a fatal bile duct cancer, is a major public health concern in areas endemic for this parasite. We report here whole-exome sequencing of eight O. viverrini–related tumors and matched normal tissue. We identified and validated 206 somatic mutations in 187 genes using Sanger sequencing and selected 15 genes for mutation prevalence screening in an additional 46 individuals with CCA (cases). In addition to the known cancer-related genes TP53 (mutated in 44.4% of cases), KRAS (16.7%) and SMAD4 (16.7%), we identified somatic mutations in 10 newly implicated genes in 14.8–3.7% of cases. These included inactivating mutations in MLL3 (in 14.8% of cases), ROBO2 (9.3%), RNF43 (9.3%) and PEG3 (5.6%), and activating mutations in the GNAS oncogene (9.3%). These genes have functions that can be broadly grouped into three biological classes: (i) deactivation of histone modifiers, (ii) activation of G protein signaling and (iii) loss of genome stability. This study provides insight into the mutational landscape contributing to O. viverrini–related CCA.

For a long time, it has been assumed that the only role of sperm at fertilization is to introduce the male genome into the egg. Recently, ideas have emerged that the epigenetic state of the sperm nucleus could influence transcription in the embryo. However, conflicting reports have challenged the existence of epigenetic marks on sperm genes, and there are no functional tests supporting the role of sperm epigenetic marking on embryonic gene expression. Here, we show that sperm is epigenetically programmed to regulate embryonic gene expression. By comparing the development of sperm- and spermatid-derived frog embryos, we show that the programming of sperm for successful development relates to its ability to regulate transcription of a set of developmentally important genes. During spermatid maturation into sperm, these genes lose H3K4me2/3 and retain H3K27me3 marks. Experimental removal of these epigenetic marks at fertilization de-regulates gene expression in the resulting embryos in a paternal chromatin-dependent manner. This demonstrates that epigenetic instructions delivered by the sperm at fertilization are required for correct regulation of gene expression in the future embryos. The epigenetic mechanisms of developmental programming revealed here are likely to relate to the mechanisms involved in transgenerational transmission of acquired traits. Understanding how parental experience can influence development of the progeny has broad potential for improving human health.

Introduction and objectives Regeneration of healthy lung tissue in patients with end-stage respiratory disease (ESRD) would cure disease, rather than treating symptoms. For this a detailed understanding of lung development is needed and the mouse has been used extensively as an in vivo genetically-modifiable model. Differentiation and validation of human induced pluripotent stem cells (hiPSCs) is entirely based on mouse literature. The most important epithelial stem cell population in developing lungs is found in distal branching tips, and these Sox9+ lung epithelial stem cells (LESCs) generate all epithelial lineages. Our objective was to develop a self-renewing, genetically-modifiable epithelial in vitro culture system from human embryonic LESCs and differentiate them into alveolar and bronchiolar cells. Methods Human embryonic LESCs were characterised using genome-wide transcriptional analysis (RNAseq) and immunohistochemistry (5–20 post-conceptional weeks). LESCs were microdissected and self-renewing expansion in 3D organoid culture was established empirically. Using RNAseq and immunohistochemistry, we assessed the similarities between cultured and fresh LESCs. Genetic stability was evaluated by karyotyping. Organoids were differentiated in vitro, or in vivo using xenotransplantation into bleomycin-injured adult mouse lungs or kidney capsule. Gene editing was done using CRISPR-Cas9 to delete SOX9 . Results RNAseq of LESCs identified broad-scale transcriptional differences between mouse and human embryonic lung stem cells. Human LESCs were successfully expanded for over 10 months as karyotypically-stable 3D organoids using a combination of 7 signalling molecules. The LESC stem cell markers, transcriptome and organoid morphology were maintained throughout the culture period. Bronchiolar and alveolar differentiation was achieved in vitro and in vivo . Moreover, xenotransplantation of organoids into bleomycin-injured adult mouse lungs was extremely efficient. Knocking out SOX9 led to a loss-of-self-renewing phenotype. Conclusions Our novel genetically-modifiable human embryonic lung culture system enables for the first time the in vitro study of human lung development and disease modelling. We anticipate that this work will transform lung regenerative medicine by guiding the development of improved protocols for hiPSC differentiation and manipulation of adult stem cells in vivo , with benefits for patients with ESRD. Additionally, our protocol for xenotransplantation following lung injury provides the first method for efficient future cell therapy.

Eggs and oocytes have a remarkable ability to induce transcription of sperm after normal fertilization and in somatic nuclei after somatic cell nuclear transfer. This ability of eggs and oocytes is essential for normal development. Nuclear actin and actin-binding proteins have been shown to contribute to transcription, although their mode of action is elusive. Here, we find that Xenopus Wave1, previously characterized as a protein involved in actin cytoskeleton organization, is present in the oocyte nucleus and is required for efficient transcriptional reprogramming. Moreover, Wave1 knockdown in embryos results in abnormal development and defective hox gene activation. Nuclear Wave1 binds by its WHD domain to active transcription components, and this binding contributes to the action of RNA polymerase II. We identify Wave1 as a maternal reprogramming factor that also has a necessary role in gene activation in development.

Regulating the MBT It has been known for more than 30 years that a defined number of cell divisions in the frog embryo precede a crucial developmental event called the midblastula transition (MBT). Collart et al. (p. 893 , published online 1 August) now elucidate a mechanism involved in the control of the MBT. DNA replication initiation factors are titrated out during early cell divisions, which controls the elongation of the cell cycle and the onset of zygotic transcription during the MBT.

Breast phyllodes tumors are rare neoplasms which present challenges for histological classification. Microscopic features are not always predictive of clinical behavior, and scarce data exist on the prognostic role of biological markers. Our study evaluated a series of 145 phyllodes tumors diagnosed at the Department of Pathology, Singapore General Hospital between 2006 and 2009, incorporating 91 (62.8%) benign, 40 (27.6%) borderline, and 14 (9.7%) malignant phyllodes tumors. Antibodies to keratin 15 (KRT15), transcobalamin I (TCN1), and homeobox gene Hox-B13 (HOXB13) were applied to sections cut from tissue microarray blocks. KRT15 and TCN1 positivity was defined when there was reactivity of 1% or more stromal cells, while HOXB13 positivity was defined using a H-score of 100 and above. Positive immunohistochemical expression for KRT15, TCN1, and HOXB13 was seen in 21 (14.5%), 96 (66.2%), and 66 (45.5%) of tumors, respectively. Stromal expression of KRT15, TCN1, and HOXB13 was significantly correlated with tumor grade (P < 0.001, P < 0.001, P = 0.012), stromal hypercellularity (P = 0.005, P < 0.001, P = 0.023), mitotic activity (P < 0.001), and microscopic borders (P = 0.006, P < 0.001, P = 0.011). Co-expression of TCN1 and HOXB13 was seen in 21 of 91 (23.1%) benign, 18 of 40 (45.0%) borderline, and 11 of 14 (78.6%) malignant tumors, suggesting that the dual-marker panels of TCN1 and HOXB13 might be helpful in classifying borderline and malignant tumors. Although expression of TCN1 alone was present in all malignant and 34 of 40 (85.0%) borderline tumors, a combined panel with HOXB13 excluded some benign cases and was a better discriminant for a significant proportion of borderline and malignant tumors.

BACKGROUND: Snakebite is a global health issue and treatment with antivenom continues to be problematic. Brown snakes (genus Pseudonaja) are the most medically important group of Australian snakes and there is controversy over the dose of brown snake antivenom. We aimed to investigate the clinical and laboratory features of definite brown snake (Pseudonaja spp.) envenoming, and determine the dose of antivenom required. METHODS AND FINDING: This was a prospective observational study of definite brown snake envenoming from the Australian Snakebite Project (ASP) based on snake identification or specific enzyme immunoassay for Pseudonaja venom. From January 2004 to January 2012 there were 149 definite brown snake bites [median age 42 y (2-81 y); 100 males]. Systemic envenoming occurred in 136 (88%) cases. All envenomed patients developed venom induced consumption coagulopathy (VICC), with complete VICC in 109 (80%) and partial VICC in 27 (20%). Systemic symptoms occurred in 61 (45%) and mild neurotoxicity in 2 (1%). Myotoxicity did not occur. Severe envenoming occurred in 51 patients (38%) and was characterised by collapse or hypotension (37), thrombotic microangiopathy (15), major haemorrhage (5), cardiac arrest (7) and death (6). The median peak venom concentration in 118 envenomed patients was 1.6 ng/mL (Range: 0.15-210 ng/mL). The median initial antivenom dose was 2 vials (Range: 1-40) in 128 patients receiving antivenom. There was no difference in INR recovery or clinical outcome between patients receiving one or more than one vial of antivenom. Free venom was not detected in 112/115 patients post-antivenom with only low concentrations (0.4 to 0.9 ng/ml) in three patients. CONCLUSIONS: Envenoming by brown snakes causes VICC and over a third of patients had serious complications including major haemorrhage, collapse and microangiopathy. The results of this study support accumulating evidence that giving more than one vial of antivenom is unnecessary in brown snake envenoming.

Abstract 2658 Background: Peripheral T-cell lymphomas (PTCL) carry a poorer prognosis compared to their B-cell counterparts and the molecular pathogenesis of PTCL is still largely unknown. The aims of this study are to characterize the molecular signatures and identify signaling pathways involved in the different subsets of PTCL and NKTCL. Materials and Methods: RNA was extracted from tumors of 60 patients with newly diagnosed PTCL and NKTCL: 21 with angioimmunoblastic T-cell lymphoma (AITL), 12 anaplastic large-cell lymphoma (ALCL), 15 peripheral T-cell lymphoma not-otherwise-specified (PTCL-NOS) and 12 with natural-killer T-cell lymphoma (NKTCL). Comparisons were made using published gene expression data files of normal T and NK T-cells. Gene expression profiling was performed using the Affymetrix HG-U133 Plus 2.0 GeneChip platform. Results: The Affymetrix expression profiling distinguishes the 48 PTCL samples from normal T-cell controls (p Discussion: Gene expression profiling identifies distinct molecular pathways in PTCL. In particular, the JAK-STAT pathway is upregulated in AITL and the NFκB pathway is dysregulated in PTCL and NKCL. Our results suggest that targeting the key kinases in these pathways may be effective in the treatment for this subset of lymphomas with poor prognosis. Disclosures: Tan: Janssen: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Research Funding.

Background Well differentiated papillary mesothelioma of the peritoneum (WDPMP) is a rare variant of epithelial mesothelioma of low malignancy potential, usually found in women with no history of asbestos exposure. In this study, we perform the first exome sequencing of WDPMP. Results WDPMP exome sequencing reveals the first somatic mutation of E2F1, R166H, to be identified in human cancer. The location is in the evolutionarily conserved DNA binding domain and computationally predicted to be mutated in the critical contact point between E2F1 and its DNA target. We show that the R166H mutation abrogates E2F1's DNA binding ability and is associated with reduced activation of E2F1 downstream target genes. Mutant E2F1 proteins are also observed in higher quantities when compared with wild-type E2F1 protein levels and the mutant protein's resistance to degradation was found to be the cause of its accumulation within mutant over-expressing cells. Cells over-expressing wild-type E2F1 show decreased proliferation compared to mutant over-expressing cells, but cell proliferation rates of mutant over-expressing cells were comparable to cells over-expressing the empty vector. Conclusions The R166H mutation in E2F1 is shown to have a deleterious effect on its DNA binding ability as well as increasing its stability and subsequent accumulation in R166H mutant cells. Based on the results, two compatible theories can be formed: R166H mutation appears to allow for protein over-expression while minimizing the apoptotic consequence and the R166H mutation may behave similarly to SV40 large T antigen, inhibiting tumor suppressive functions of retinoblastoma protein 1.

A management strategy for the imported fire ants, Solenopsis invicta Buren and S. richteri Forel, presents conceptual and implemental problems. Management strategies for social insects other than beneficial species have not yet been achieved. Such strategies are assumed to be possible since populations of social insects, like those of nonsocial insects, are subject to natural biotic and abiotic constraints. The work of Way (1953) in Tanzania, and of Room (1971), Majer (1972), and Leston (l973) in Ghana, indicate that ant populations can be increased or reduced by manipulating various ecological factors. There is also a considerable body of information by European workers concerning their attempts to translocate and enhance populations of various ant species of the Formica rufa Linne group as protective biological control agents against forest insect pests. Thus background information which might be helpful in the development of management strategies for social insects such as fire ants is available.

A 17-day trip was made through the States of Sao Paulo, Mato Grosso, Rondania Territory, Goias, and Minas Gerais, Brazil, to study the Solenopsis saevissima (Smith) complex. Special emphasis was on the distribution and ecology of Solenopsis invicta Buren. S. invicta was established in an area bordered by Caceres, Rondonopolis, Chapada, and Terenos in the State of Mato Grosso, and was also found at Porto Velho, Rondonia Territory. Active colonies were present only in moist or protected areas. This study reconfirmed a previous postulation that areas of Mato Grosso were a part of the homeland of S. invicta , the “red imported fire ant” of southeastern United States.

Developmental data on the velvetbean caterpillar (VRC), collected in both controlled-environment and field settings, allowed the computation of parameters depicting the temperature-dependent development of VBC. These parameters were associated with a previously derived poikilothermic developmental model and were derived to allow computation of the distribution of stage-specific emergence times for the VBC, given any particular temperature regime. These evaluations elucidate several problems (e.g., mortality) associated with attempts to validate single processes (e.g., development) in complex field environments.

The history of Spicaria rileyi (Farlow) Charles and Spicaria prasina (Maub.) Sawada is discussed. The genus Nomuraea is resurrected and Spicaria rileyi is transferred to that genus. Spicaria prasina is placed in synonomy with Nomuraea rileyi (Farlow) Samson.

Effects of temperature on velvetbean caterpillar (VBC), Anticarsia gemmatalis HA¼bner (Lepidoptera: Noctuidae), oviposition, egg hatch, and adult longevity were investigated at 3 regimes: (1) constant temperatures of 21.1°, 23.9°, 26.7°, 29.4°, and 32.2°C (on artificial diet); (2) variable temperatures simulating “summer” at Gainesville, FL (artificial diet and soybean foliage); (3) field-reared adults (in cages on soybean). Mean oviposition was highest at 26.7°C (842.2 egg/female) and lowest at 32.2° (310.0 eggs/female) constant. Peak average daily oviposition was higher for young females and was delayed as temperatures decreased. Mean egg hatch was high initially but decreased with maternal age. Female longevity varied from a mean of 24.8 days (21.1°C) to 11.2 days (32.2°C). Unmated females lived significantly (I± = .05) longer than mated females at a given temperature.

Effects of soybean, Glycine max (L.) cv. Bragg, phenology on leaf consumption and development of the velvetbean caterpillar (VBC), Anticarsia gemmatalis Hubner, were investigated at 26.7 ± 1° C, 14L:10D photoperiod, and RH > 80%. Soybean plant stages were designated as early vegetative (V2–V5), late vegetative (V5–V8), flowering (R1–R2), pod set to pod fill (R4–R6), and senescent (R7–R8). Mean leaf fresh weight consumed by 4th- and 5th-instar VBC larvae declined as plant phenology progressed. Dry-weight consumption did not tend to decline with plant phenology and was more uniform for all plant growth stages than the freshweight values. Mean larval development time varied from 12.6 to 14.3 days, and mean time from egg hatch to adult varied from 21.9 to 23.4 days when larvae were fed late-vegatative and senescent foliage, respectively. Larvae fed senescent leaves produced pupae with reduced weight. Sex ratio (male/female) varied from 0.83 (flowering stage) to 1.37 (late-vegetative stage).

Different concentrations of nuclear polyhedrosis virus (NPV) and insecticides (carbaryl and methomyl) were applied to natural infestations of the velvetbean caterpillar (VBC), Anticarsia gemmatalis Hubner, on soybean. Their efficacy in reducing VBC larval populations and their effect on naturally occurring epizootics of the entomogenous fungus Nomuraea rileyi (Farlow) Samson were determined. Persistence of the virus in the field was measured. Virus application rates of 75, 145, and 287.5 larval equivalents (LE)/ha suppressed VBC populations below damaging levels. Most virus-induced mortality occurred at 6, 7, and 8 days after spraying. The virus retained 65 to 70% of its original pathogenicity 7 days after treatment and 25 to 35% at 10 days. All virus doses greatly reduced the magnitude of a N. rileyi epizootic. Carbaryl or methomyl applied once at 0.28 or 0.07 kg AI/ha effectively suppressed VBC populations below economic levels for the remainder of the season. High concentrations of insecticides reduced the impact of N. rileyi by reducing the natural substrate, VBC larvae.

High pupal mortality experienced during laboratory rearing of Biosteres longicaudatus , a parasitoid of the Caribbean fruit fly Anastrepha suspensa was attributed primarily to the action of two species of opportunistic pathogens, Serratia marcescens and Pseudomonas aeruginosa . These bacteria were best able to overwhelm both parasitized and nonparasitized fly larvae and pupae when they were subjected to thermal stress (rearing temperatures >30°C). Methenamine mandelate chemotherapy had no prophylactic effect, but potentially deleterious side effects (aberrant fly premating sounds) were caused by incorporation of this antibiotic in the A. suspensa larval rearing medium. Control was effected by optimizing the cultural conditions rather than by the use of antibiotics.

Burenella dimorpha infects the tropical fire ant, Solenopsis geminata , producing two morphologically distinct types of spores. A binucleate, nonpansporoblast membrane-bounded (NPMB) spore develops in and destroys the hypodermis, rupturing the cuticle in the pupal stage. A uninucleate, pansporoblast membrane-bounded (PMB) spore develops in the fat body. Adult ants cannibalize ruptured pupae but do not ingest spores. Instead, the spores and particulate foods are diverted to the infrabuccal cavity, formed into an infrabuccal pellet, and fed to fourth-instar larvae only. This larval instar is the only stage in the life cycle of S. geminata that is vulnerable to infection. NPMB spores are infective, but PMB spores do not extrude their polar filaments in the larval gut and are expelled in the meconium upon pupation.

A new species of Microsporida, Thelohania solenopsae , is described from the red imported fire ant, Solenopsis invicta . The Thelohania infections are localized in the fat body of workers. Meronts causing infections of progeny are found in the ovaries of queens. Spores occur only in adult ants and only vegetative stages are present in larvae and pupae. Both uninucleate octospores (eight spores within a pansporoblast membrane) and binucleate free spores (spores developing in isolation) are formed.

Significant differences in infection rates of the entomogenous fungus, Nomuraea rileyi (Farlow) Samson among populations of Anticarsia gemmatalis (Hubner) in soybean treated with benomyl, benomyl and methyl parathion, and benomyl and carbaryl were observed. Development of N. rileyi was delayed in treated plots for at least 3 weeks. The yield of the check plot was significantly higher than that of the pesticide treated plots.

The inter-instar susceptibility of the velvetbean caterpillar to a nucleopolyhedrosis virus was quantitated. Analysis of response curves revealed a 930-fold range in susceptibility between the most sensitive and the most resistent individuals in these larval populations. The LD50 values for 2nd, 3rd, 4th, and 5th instars were 103, 432, 682, and 4100 polyhedra/larva, respectively. At 26.7°C the virus replicated efficiently in all larval instars. Virus replication was slower at lower temperatures (15.6°C, 21.1°C) but the cumulative virus mortality at these these temperatures were similar fo that achieved at 26.7°C. Incubation at higher temperatures (32.2°C), however, caused a 30–50% less virus mortality than occurred at lower temperatures.

A randomized small plot test was used to compare the effectiveness of a nuclear-polyhedrosis virus and toxaphene-DDT, a standard insecticide for Heliothis control on cotton. There was no significant difference in Heliothis control between the four virus treatments of 1.2 × 10 11 , 3.0 × 10 11 , 6.0 × 10 11 , and 18.0 × 10 11 polyhedral inclusion bodies (PIB)/acre. All four virus treatments compared favorably with the insecticide in controlling Heliothis zea or Heliothis virescens on cotton.

A second work conference was called by the Organization Committee for Future Meetings to discuss and review the present status of research, registration, and standardization of the nuclear-polyhedrosis virus of Heliothis . The meeting was sponsored by the Department of Entomology and held on the campus of Mississippi State University on March 29 and 30, 1967. Attending were 51 persons from the U. S. Department of Agriculture, Food and Drug Administration, private industries, several university and State experiment stations, and consulting entomologists.

A microsporidan, Thelohania sp., was isolated from workers of the black imported fire ant, Solenopsis richteri Forel, in Montevideo, Uruguay, and Las Flores, Argentina, and from a Solenopsis sp. in Montevideo, in March and May of 1974. Many of the colonies exhibiting high levels of infection in March were abandoned or drastically reduced in size when re-examined in May. No disease was observed in populations of S. blumi Buren or S. quinquecuspis Forel.

A method was developed for determining the number of cadavers of the velvetbean caterpillar, Anticarsia gemmatalis (Hubner), infected with the fungal pathogen Nomuraea rileyi (Farlow) Samson in the developmental stage just prior to conidia formation (white stage) in a soybean field. One-third of the total number of cadavers in this developmental stage were observed to fall on the shake cloth during population sampling; (approximately two-thirds remained on the plant). Estimates of white cadaver counts from laboratory-held lots of field-collected larvae did not correlate with actual field counts.

Reinfectivity studies were conducted on late instar lovebug larvae, Plecia nearctica Hardy (Diptera-Bibionidae), inoculated with Tolypocladium cylindrosporum Gams, Metarhizium anisopliae (Metch.) Sor. and Beauveria bassiana (Bals.) Vuill. which were isolated and identified from dead larvae. Two tests demonstrated that each fungus apparently affected larval mortality; however, data analyses indicated that only B. bassiana caused significant mortality levels (27% to 33%). Problems related to reinfectivity tests are discussed. Nine additional fungi are reported from dead and moribund larvae collected in the field.

Populations of Plecia nearctica Hardy were surveyed during the spring and fall of 1973 to monitor adult flights which usually occur in May and September. The May adult flight was noticeably smaller. A large flight was observed in September. During early spring, 5 species of fungi were isolated and identified from dead and moribund lovebug larvae collected in the Gainesville area. In the fall, 2 additional fungal species were isolated, 1 from adults during the September flight and 1 from laboratory reared larvae.