Methoprene modulates the effect of diet on male melon fly,Bactrocera cucurbitae,performance at mating aggregations

The effect of access to dietary protein (P) (hydrolyzed yeast) and/or treatment with a juvenile hormone analogue, methoprene (M), (in addition to sugar and water) on male aggregation (lekking) behaviour and mating success was studied in a laboratory strain of the melon fly, Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae). Six‐day‐old males were treated with (1) protein and methoprene (M+P+), (2) only protein (M?P+), or (3) only methoprene (M+P?), and compared with 14‐day‐old sexually mature untreated males (M?P?). The lekking behaviour of the four groups of males when competing for virgin sexually mature females (14 –16 days old) was observed in field cages. The following parameters were measured at male aggregations: lek initiation, lek participation, males calling, male–male interaction, female acceptance index, and mating success. For all these parameters, the M+P+ males significantly outperformed the other males. Moreover, for all parameters, there was a similar trend with M+P+ > M?P+ > M?P? > M+P?. More M+P+ males called and initiated and participated in lek activities than all other types of male, which resulted in higher mating success. They had also fewer unsuccessful copulation attempts than their counterparts. Whereas treatment with methoprene alone had a negative effect in young males with only access to sugar, access to dietary protein alone significantly improved young male sexual performance; moreover, the provision of methoprene together with protein had a synergistic effect, improving further male performance at leks. The results are of great relevance for enhancing the application of the sterile insect technique (SIT) against this pest species. The fact that access to dietary protein and treatment of sterile males with methoprene improves mating success means that SIT cost‐effectiveness is increased, as more released males survive to sexual maturity.     

Application of the most probable number method to estimations of entomopathogenic nematode populations in soil

The calculation of most probable numbers (mpn) was used for the estimation of numbers of infective entomopathogenic nematodes in soil. The mpn concept was first introduced in bacteriology as a means of estimating numbers of organisms in a substrate without a direct count, in cases where direct enumeration could not be applied. In the work reported here, soil samples infested with infective juveniles (IJs) of Heterorhabditis megidis (isolate HF85) were diluted with uninfested soil and the diluted soils were baited with mealworm larvae (Tenebrio molitor). The mpn of infective units of IJs in the undiluted soil was calculated. The mpn calculation was found to be applicable to entomopathogenic nematodes in soil under particular conditions. It could be successfully applied to data for the response of soil units but not to the data based on the response of individual insects, as the latter did not confirm to a Poisson series. The calculated mpn represented between 2.9 and 7.1% of the initial inoculum of IJs. It was suggested that IJs might act as a group in infecting an insect host. Using the data for Tenebrio mortality on parasitisation, the mpn based on quantal response of the mealworms would therefore not give the true density of IJs in the soil sample but the effective density, or the quantity of infective units. Although the biological significance of the infective unit needs further clarification, mpn was found to be a useful parameter for use in comparative experiments.     

Reduced generation time of apple seedlings to within a year by means of a plant virus vector: a new plant‐breeding technique with no transmission of genetic modification to the next generation

Fruit trees have a long juvenile phase. For example, the juvenile phase of apple (Malus × domestica) generally lasts for 5–12 years and is a serious constraint for genetic analysis and for creating new apple cultivars through cross‐breeding. If modification of the genes involved in the transition from the juvenile phase to the adult phase can enable apple to complete its life cycle within 1 year, as seen in herbaceous plants, a significant enhancement in apple breeding will be realized. Here, we report a novel technology that simultaneously promotes expression of Arabidopsis FLOWERING LOCUS T gene (AtFT) and silencing of apple TERMINAL FLOWER 1 gene (MdTFL1‐1) using an Apple latent spherical virus (ALSV) vector (ALSV‐AtFT/MdTFL1) to accelerate flowering time and life cycle in apple seedlings. When apple cotyledons were inoculated with ALSV‐AtFT/MdTFL1 immediately after germination, more than 90% of infected seedlings started flowering within 1.5–3 months, and almost all early‐flowering seedlings continuously produced flower buds on the lateral and axillary shoots. Cross‐pollination between early‐flowering apple plants produced fruits with seeds, indicating that ALSV‐AtFT/MdTFL1 inoculation successfully reduced the time required for completion of the apple life cycle to 1 year or less. Apple latent spherical virus was not transmitted via seeds to successive progenies in most cases, and thus, this method will serve as a new breeding technique that does not pass genetic modification to the next generation.     

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases ^1-7. Progression of tumors from a benign to a metastatic state is a complex event ⁸ and has been modeled in Drosophila to help us better understand the genetic basis of this disease ⁹. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system ¹⁰ and exploits the cooperation between an activated oncogene, Ras^V12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors ⁹. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.     

Localization and characterization of T-cell subpopulations and natural killer cells (HNK 1+ cells) in the human tonsilla palatina

Summary In the present study attention was focussed on several lymphoid subpopulations and specific stationary cells of the human tonsilla palatina. They were labeled at the light- and electron-microscopic levels by means of monoclonal antibodies to cell surface antigens. Cells resembling interdigitating cells (IDC-like cells) within the crypt epithelium and the interdigitating cells in the parafollicular T-cell region express the HLA-DR antigen. This fact suggests a relationship between these two populations of cells. Both cell types were frequently found in close contact to T-helper cells labeled with Anti-Leu 3a. This fact is discussed as a confirmation of earlier suggestions that the tonsillar crypt epithelium serves as T-cell region. Cytotoxic/ suppressor-T cells (OKT 8 ⁺) and Leu 7-positive cells do not appear to contact interdigitating cells. Anti-Leu 7 is a monoclonal antibody, that defines a differentiation antigen shown to be selectively expressed on human natural killer cells (NK-cells). With the use of the immuno-electron-microscopic labeling method it was possible to analyze the ultrastructure of this lymphoid subpopulation. Two morphologically distinguishable subtypes of Leu 7-positive cells populate different microenvironments: The Leu 7-positive large-granular lymphocyte was predominantly found in the crypt epithelium, while numerous Leu 7-positive cells located in the germinal centers had the appearance of small lymphocytes. This finding is discussed in favour of distinct phenotypes representing different stages in a differentiation pathway of the maturing NK-cell: Small Leu 7-positive lymphocytes in the germinal centers are supposed to be functionally inactive precursors, and only the Leu 7-positive large granulated lymphocytes in the crypt epithelium may represent differentiated active NK-cells. This interpretation is in agreement with the observation that the tonsilla palatina, in spite of containing numerous Leu 7-positive cells, shows only low NK-activity against tumor cells.Glossary of Abbreviations used in this Paper DAB diamino-benzidine - DMSO dimethyl sulfoxide - HLA human leucocyte antigen - HLA-DR human leucocyte antigen, D-region related - Ia-antigen immune-associated antigen of the MHC - IDC interdigitating cell - IDC-like cell cell that resembles an interdigitating cell - LGL large granular lymphocyte - MHC major histocompatibility gene complex - NK-cell natural killer cell - PBS phosphate-buffered saline     

Inbreeding and Hybridizing Cyst Nematodes on Pruned Soybeans in Petri Plates

Inbred nematodes propagated on a selecting host are likely to have homozygous genes of interest for investigating the genetics of host-parasite associations. A technique is presented to inbreed soybean cyst nematodes, by sibling matings at each generation, and to cross inbred lines. Soybean seedlings with severely trimmed cotyledons survive well on 0.8% agar. Eggs from a single female are incubated in water in a microtiter well. Virgin as well as mated females result from inoculation of two juveniles per root. Sibling males from the same source are produced by mass inoculations of eggs. Males are added individually to unmated females. Overall success for fertile females was 14% in 1,368 isolations. Three generations of inbreeding by siblings were achieved using nematodes from two populations that differ in their ability to reproduce on differential soybeans. Hybrids from crosses of the two inbred lines tested on differential hosts showed that the influence of Population 1 (selected and inbred on PI 209332) is greater than that of Population 2 (selected and inbred on PI 89772). Reciprocal crosses suggest that the influence of males is stronger than that of females in determining host specificity of F₁ offspring in these crosses. Our technique is simple and effective for inbreeding and crossing soybean cyst nematodes.     

Immunohistochemical characterization of the thymic microenvironment

Summary The epithelial framework of the human thymus has been studied in parallel by immunohistochemical methods at the light- and electron-microscopic levels. Different monoclonal antibodies were used, reacting with components of the major histocompatibility complex, keratins, thymic hormones and other as yet antigenically undefined substances, which show specific immunoreactivities with human thymus epithelial cells.The electron-microscopic immunocytochemical observations clearly confirm microtopographical differences of epithelial cells not only between the thymic cortex and medulla, but also within the cortex itself. At least four subtypes of epithelial cells could be distinguished: 1) the cortical surface epithelium; 2) the main cortical epithelial cells and thymic nurse cells; 3) the medullary epithelial cells; and 4) the epithelial cells of Hassall's corpuscles.The various epithelial cell types of the thymus display several common features like tonofilaments, desmosomes and some surface antigens as demonstrated by anti-KiM3. In other respects, however, they differ from each other. The cortical subtype of thymic epithelial cells including the thymic nurse cells shows a distinct pattern of surface antigens reacting positively with antibodies against HLA-DR (anti-HLA-DR) and anti-21A62E. Electron-microscopic immunocytochemistry with these antibodies clearly reveals a surface labeling and a narrow contact to cortical thymocytes particularly in the peripheral cortical regions. An alternative staining pattern is realized by antibodies to some antigens associated with other subtypes of thymic epithelial cells. Medullary epithelial cells as well as the cortical surface epithelium react likewise positively with antibodies to special surface antigens (anti-Ep-1), to special epitopes of cytokeratin (anti-IV/82), and to thymic hormones (anti-FTS). The functional significance of distinct microenvironments within the thymus provided by different epithelial cells is discussed in view of the maturation of T-precursor cells.Glossary of Abbreviations Anti-X anti-X antibody - APUD-cells amine precursor uptake and decarboxylation (gastro-intestinal endocrine cells) - DAB diamino-benzidine - DMSO dimethyl sulfoxide - FTS facteur thymique sérique - HLA-A, B, C human leucocyte antigen, A, B, C-region related - HLA-DR human leucocyte antigen, D-region related - IDC interdigitating cell - MHC major histocompatibility gene complex - PBS phosphate-buffered saline - TNC thymic nurse cell This investigation was supported by grants from the Deutsche Forschungsgemeinschaft, and its Sonderforschungsbereich 111Fellow of the Alexander von Humbold-Stiftung, Institute of Pathology, University of Würzburg, Federal Republic of GermanyThe authors appreciate the contribution of human thymus tissue from Professor Alexander Bernhard, Abteilung kardiovasculäre Chirurgie der Universität Kiel; the gift of monoclonal antibodies from Dr. M.J.D. Anderson, Dr. M. Dardenne and Dr. H.J. Radzun; and the excellent technical assistence of Mrs. O.M. Bracker, Mrs. H. Hansen, Mrs. R. Köpke, Mrs. M. v. Kolszynski, Mrs. J. Quitzau, Mrs. H. Siebke, and Mrs. H. Waluk     

Non-lethal rapid biodiversity assessment

For several animal taxa, non-lethal techniques that do not rely on collecting individuals are routinely used to assess biodiversity (e.g. point counts in birds). Identification often relies on the ability of the observer, are subjected to errors, but populations are not impacted. Thus, multiple counting sessions (MCS) that allow using robust analyses (e.g. unbiased Chao richness estimate) are available. However, for most species (e.g. arthropods), trap systems must be set up. Killed individuals are collected and later accurately identified in the laboratory, but unbiased MCS become unavailable. Environmental DNA bar-coding provides an alternative, yet it requires important technical support and is not designed for MCS. Lethal rapid biodiversity assessments (RBA), derived from classical trap surveys and based on less accurate identifications (morphospecies are used), have been successfully developed to relax technical constraints. In this study, we combined non-lethal and RBA approaches to address logistical, analytical and ethical issues. We tested five versions of a protocol to visually survey the macro-fauna of hedgerows. A large number of individuals were directly identified in the field, mostly arthropods but also vertebrates. Identification error varied with taxonomic level and lineage, but remained low at the morphospecies level. Importantly, estimates tended to reach asymptotes, suggesting that local richness was appropriately appraised. Like any technique, non-lethal RBA (NL-RBA) present both advantages and weaknesses, and may improve the toolbox to survey biodiversity.     

Methods for Estimating the Parameters of Nonlinear Adsorption Isotherms of Langmuir and Freundlich Types from a Response Curve of Pulse Input of an Adsorbate

Methods for estimating the parameters of nonlinear adsorption isotherms of Langmuir and Freundlich types from a pulse response curve are proposed here based on the migration rate of an adsorbate at a constant concentration and the mean residence time of the adsorbate in a bed. The methods were used to estimate the parameters in isotherms for various combinations of adsorbent and adsorbate. The isotherms estimated by the proposed methods were compared with those estimated by conventional methods. It was demonstrated that the proposed methods could evaluate the parameters with fairly good precision when the type of isotherm was known. The criteria for discriminating the type of isotherm from the pulse response curve are also described.     

Apparent digestibility of broken rice in horses using in vivo and in vitro methods

The aim of this study was to assess the apparent digestibility of broken rice using total collection of feces and the pepsin-cellulase in vitro technique to provide updated and more accurate digestion coefficients for this by-product when fed to horses. The in vivo digestibility trial was consecutively performed, using five adult geldings, weighing 555.6 kg on average. First, hay was given as the only feedstuff, while second, the experimental diet consisted of the same hay plus broken rice at a forage-to-concentrate ratio of 70/30 (on dry matter (DM) basis). Feces were collected over 6 days preceded by a 14-day adaptation period. The digestibility trial was carried out to determine the digestion coefficients for DM, organic matter (OM), CP and gross energy in both diets, while apparent digestion coefficients for the same parameters were calculated for broken rice alone, using the difference between the two sets of results. At the same time, an in vitro trial was carried out using pepsin-cellulase technique on the samples of hay and broken rice tested during the in vivo trial. As expected, supplementation with broken rice increased digestibility according to all the parameters used. The high OM digestion coefficients of broken rice were confirmed both by the calculated in vivo method and by the predicted results of pepsin-cellulase technique (92.6% and 87.1%, respectively), underlining the high digestibility of this by-product when fed to horses.