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Curricular guidelines promote standardized approaches to coverage of essential knowledge and skills in undergraduate education. The American Society for Microbiology (ASM) Curriculum Guidelines for Undergraduate Microbiology were developed in 2012. Continuous, rapid growth of knowledge in science and a dynamic, changing world necessitate updates to these guidelines. As such, ASM formed a task force in the summer of 2022. The task force assessed the 2012 ASM Curriculum Guidelines considering advancements in technology, an understanding of an expanded role of microbes, and a broader scope addressing relevant social and environmental aspects of microbiology. Language in the updated guidelines was also modified to better include eukaryotic microbes, viruses, and other acellular microbes. The task force formed working groups, each aimed at revising specific sections of the 2012 ASM Curriculum Guidelines. The revisions to the ASM Curriculum Guidelines were reviewed by subject matter experts and education stakeholders. Feedback from this peer review was incorporated into the updated guidelines, and further comments were solicited from the ASM Conference of Undergraduate Educators (ASMCUE) attendees in November 2023 before these guidelines were finalized. In this article, we describe the rationale and development of updated ASM Curriculum Guidelines which identify foundational concepts that will serve to improve microbial literacy and that can be expanded upon to address more advanced and specialized topics.

This guideline has been prepared by the ACPSEM to provide a standardised quality assurance program to be used within General X-ray imaging environments. The guideline includes the responsibilities of various multidisciplinary team members within medical imaging facilities. It must be noted that the listed tests and testing frequencies are not intended to replace or become regulatory requirements. Implementing a quality assurance program as outlined in this position paper is there to ensure best practice for imaging facilities by providing a framework to establish and monitor correct equipment performance. The current document has been produced through an extensive review of current international practices and local experience within the Australasian healthcare environment. Due to the constant evolution of digital radiographic equipment, there is no current consensus in international quality assurance guidelines as they continue to be adapted and updated. This document describes the current state of the use of digital General X-ray equipment in the Australasian environment and provides recommendations of test procedures that may be best suited for the current medical imaging climate in Australasia. Due to the everchanging developments in the medical imaging environment and the ability of new technologies to perform more complex tasks it is believed that in the future this document will be further reviewed in the hopes of producing a more globally agreed upon standard quality assurance program. Any such adjustments that are deemed to be necessary to Version 1.0 of this document will be provided in electronic format on the ACPSEM website with a notification to all parties involved in the use of digital General X-ray equipment. This guideline does not provide detailed methodologies for all the quality control tests recommended as it is it is expected that the professionals implementing aspects of this quality assurance program have the working knowledge and access to appropriate resources to develop testing methodologies appropriate for their local imaging environment., (© 2024. The Author(s).)

Like other lepidopteran insects, males of the tobacco cutworm moth, Spodoptera litura produce two kinds of spermatozoa, eupyrene (nucleate) and apyrene (anucleate) sperm. Formed in the testis, both kinds of sperm are released into the male reproductive tract in an immature form and are stored in the duplex region of the tract. Neither type of sperm is motile at this stage. When stored apyrene sperm from the duplex are treated in vitro with an extract of the prostatic region of the male tract, or with mammalian trypsin, they become motile; activation is greater and achieved more rapidly with increasing concentration of extract or enzyme. The activating effect of prostatic extract is blocked by soybean trypsin inhibitor (SBTI), also in a dose-dependent way. These results suggest that the normal sperm-activating process is due to an endogenous trypsin-like protease produced in the prostatic region. Proteomic analysis of S. litura prostatic extracts revealed a Trypsin-Like Serine Protease, TLSP, molecular weight 27 kDa, whose 199-residue amino acid sequence is identical to that of a predicted protein from the S. litura genome and is highly similar to predicted proteins encoded by genes in the genomes of several other noctuid moth species. Surprisingly, TLSP is only distantly related to Serine Protease 2 (initiatorin) of the silkmoth, Bombyx mori, the only identified lepidopteran protein so far shown to activate sperm. TLSP has features typical of secreted proteins, probably being synthesized as an inactive precursor zymogen, which is later activated by proteolytic cleavage. cDNA was synthesized from total RNA extracted from the prostatic region and was used to examine TLSP expression using qPCR. tlsp mRNA was expressed in both the prostatic region and the accessory glands of the male tract. Injection of TLSP-specific dsRNA into adult males caused a significant reduction after 24 h in tlsp mRNA levels in both locations. The number of eggs laid by females mated to adult males that were given TLSP dsRNA in 10 % honey solution, and the fertility (% hatched) of the eggs were reduced. Injecting pupae with TLSP dsRNA caused the later activation of apyrene sperm motility by adult male prostatic extracts to be significantly reduced compared to controls. Exposure of S. litura pupae to ionizing radiation significantly reduced expression of tlsp mRNA in the prostatic part and accessory gland of irradiated males in both the irradiated generation and also in their (unirradiated) F1 progeny. The implications of these findings for the use of the inherited sterility technique for the control of S. litura and other pest Lepidoptera are discussed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Lepidoptera are unusual in possessing two distinct kinds of sperm, regular nucleated (eupyrene) sperm and anucleate (apyrene) sperm ('parasperm'). Sperm of both types are transferred to the female and are required for male fertility. Apyrene sperm play 'helper' roles, assisting eupyrene sperm to gain access to unfertilized eggs and influencing the reproductive behavior of mated female moths. Sperm development and behavior are promising targets for environmentally safer, target-specific biorational control strategies in lepidopteran pest insects. Sperm dimorphism provides a wide window in which to manipulate sperm functionality and dynamics, thereby impairing the reproductive fitness of pest species. Opportunities to interfere with spermatozoa are available not only while sperm are still in the male (before copulation), but also in the female (after copulation, when sperm are still in the male-provided spermatophore, or during storage in the female's spermatheca). Biomolecular technologies like RNAi, miRNAs and CRISPR-Cas9 are promising strategies to achieve lepidopteran pest control by targeting genes directly or indirectly involved in dichotomous sperm production, function, or persistence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Seth, Yadav and Reynolds.)

Competing Interests: The author declares no competing interest.

In Washington State, like many states, there is a shortage of forensically trained mental health clinicians to work with criminal justice-involved individuals. At the direction of the state legislature, a collaborative project was undertaken by the University of Washington, the state Department of Social and Health Services, and a state psychiatric hospital to develop a proposal for a jointly sponsored forensic teaching service. The authors reviewed the literature, surveyed and interviewed forensic psychiatry and psychology training directors, and conducted site visits of selected training programs that offer multidisciplinary training or have affiliations with state hospitals. The authors conducted focus groups of additional stakeholders, including clinicians and patients in forensic settings, to better understand the needs in Washington. The authors report on several common benefits and barriers to establishing forensic teaching services. Other states and forensic programs may find this article useful in identifying common considerations for forensic mental health teaching services., (© 2019 American Academy of Forensic Sciences.)

Background: Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(-NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology., Results: Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids., Conclusion: Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.

Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells., (Published by Elsevier Inc.)

Genomic and transcriptomics analyses have revealed human head and body lice to be almost genetically identical; although con-specific, they nevertheless occupy distinct ecological niches and have differing feeding patterns. Most importantly, while head lice are not known to be vector competent, body lice can transmit three serious bacterial diseases; epidemictyphus, trench fever, and relapsing fever. In order to gain insights into the molecular bases for these differences, we analyzed alternative splicing (AS) using next-generation sequencing data for one strain of head lice and one strain of body lice. We identified a total of 3,598 AS events which were head or body lice specific. Exon skipping AS events were overrepresented among both head and body lice, whereas intron retention events were underrepresented in both. However, both the enrichment of exon skipping and the underrepresentation of intron retention are significantly stronger in body lice compared with head lice. Genes containing body louse-specific AS events were found to be significantly enriched for functions associated with development of the nervous system, salivary gland, trachea, and ovarian follicle cells, as well as regulation of transcription. In contrast, no functional categories were overrepresented among genes with head louse-specific AS events. Together, our results constitute the first evidence for transcript pool differences in head and body lice, providing insights into molecular adaptations that enabled human lice to adapt to clothing, and representing a powerful illustration of the pivotal role AS can play in functional adaptation., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Most poxvirus proteins are either highly conserved and essential for basic steps in replication or less conserved and involved in host interactions. Homologs of the CPXV219 protein, encoded by cowpox virus, are present in nearly all chordopoxvirus genera and some species have multiple copies. The CPXV219 homologs have estimated masses of greater than 200 kDa, making them the largest known poxvirus proteins. We showed that CPXV219 was expressed early in infection and cleaved into N- and C-terminal fragments that remained associated. The protein has a signal peptide and transited the secretory pathway where extensive glycosylation and proteolytic cleavage occurred. CPXV219 was located by immunofluorescence microscopy in association with the endoplasmic reticulum, Golgi apparatus and plasma membrane. In non-permeabilized cells, CPXV219 was accessible to external antibody and biotinylation. Mutants that did not express CPXV219 replicated normally in cell culture and retained virulence in a mouse respiratory infection model., (Published by Elsevier Inc.)

RNA interference (RNAi) is a specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful reverse genetics tool in insects. Unfortunately, however, this technology has been limited by the variable sensitivity of insect species to RNAi. We propose that rapid degradation of dsRNA in insect hemolymph could impede gene silencing by RNAi and experimentally investigate the dynamics of dsRNA persistence in two insects, the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. An ex vivo assay revealed that dsRNA was rapidly degraded by an enzyme in M. sexta hemolymph plasma, whilst dsRNA persisted much longer in B. germanica plasma. A quantitative reverse transcription PCR-based assay revealed that dsRNA, accordingly, disappeared rapidly from M. sexta hemolymph in vivo. The M. sexta dsRNAse is inactivated by exposure to high temperature and is inhibited by EDTA. These findings lead us to propose that the rate of persistence of dsRNA in insect hemolymph (mediated by the action of one or more nucleases) could be an important factor in determining the susceptibility of insect species to RNAi., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Gene silencing by RNA interference (RNAi) can be a useful reverse genetics tool in eukaryotes. However, some species appear refractory to RNAi. To study the role of the differential expression of RNAi proteins in RNAi, we isolated partial dicer-2, argonaute-2 translin, vasa intronic gene (VIG) and tudor staphylococcus/micrococcal nuclease (TSN) genes from the tobacco hornworm, Manduca sexta, a well-studied insect model which we have found to be variably sensitive to RNAi. We found that the RNAi gene, translin, was expressed at minimal levels in M. sexta tissue and that there is a specific, dose-dependent upregulation of dicer-2 and argonaute-2 expression in response to injection with dsRNA, but no upregulation of the other genes tested. Upregulation of gene expression was rapid and transient. In order to prolong the upregulation we introduced multiple doses of dsRNA, resulting in multiple peaks of dicer-2 gene expression. Our results have implications for the design of RNAi experiments and may help to explain differences in the sensitivity of eukaryotic organisms to RNAi., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This is a retrospective review of 46 primary total hip arthroplasties using a first-generation highly cross-linked ultrahigh-molecular-weight polyethylene liner (Crossfire; Stryker, Mahwah, NJ) with an average of 9-year follow-up. The purpose of this study was to measure linear penetration rate of first-generation polyethylene to determine if it maintains its wear resistance and fatigue strength over an extended period compared with conventional polyethylene. The mean (SD) total penetration was 0.339 (0.204) mm, and the mean (SD) penetration rate was 0.037 (0.022) mm/y. Our study demonstrated a 74% reduction in total penetration of highly cross-linked polyethylene when compared with historical controls using conventional polyethylene at an average of 9 years. Our results support the belief that highly cross-linked polyethylene does retain its wear resistance over an extended period., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Host innate immunity plays a central role in detecting and eliminating microbial pathogenic infections in both vertebrate and invertebrate animals. Entomopathogenic or insect pathogenic nematodes are of particular importance for the control of insect pests and vectors of pathogens, while insect-borne nematodes cause serious diseases in humans. Recent work has begun to use the power of insect models to investigate host-nematode interactions and uncover host antiparasitic immune reactions. This review describes recent findings on innate immune evasion strategies of parasitic nematodes and host cellular and humoral responses to the infection. Such information can be used to model diseases caused by human parasitic nematodes and provide clues indicating directions for research into the interplay between vector insects and their invading tropical parasites., (Published by Elsevier Ltd.)

The double-stranded DNA genome of vaccinia virus (VACV), the prototype poxvirus, contains approximately 200 open reading frames (ORFs) that are transcribed at early, intermediate, and late stages of infection. Previous high-throughput deep RNA sequencing allowed us to map 118 VACV early genes that are expressed before viral DNA replication and 93 postreplicative genes. However, the intermediate- and late-stage postreplicative genes could not be differentiated. Here, we synchronized infections with a reversible inhibitor of DNA replication and used a VACV mutant that conditionally transcribes late genes to sequence the two classes of mRNAs. In addition, each postreplicative ORF was individually expressed under conditions that distinguished intermediate and late classes. We identified 38 VACV genes that belong to the late class and 53 that belong to the intermediate class, with some of the latter continuing to be expressed late. These data allowed us to prepare a genome-wide early, intermediate, and late transcription map. Inspection of sequences upstream of these ORFs revealed distinctive characteristics of intermediate and late promoters and suggested that some promoters have intermediate and late elements. The intermediate genes encoded many DNA binding/packaging and core-associated proteins in addition to late transcription factors; the late genes encoded many morphogenesis and mature virion membrane proteins, including those involved in entry, in addition to early transcription factors. The top-ranked antigens for CD4(+) T cells and B cells were mainly intermediate rather than late gene products. The differentiation of intermediate and late genes may enhance understanding of poxvirus replication and lead to improvements in expression vectors and recombinant vaccines.

Manduca sexta allatotropin (Manse-AT) is a multifunctional neuropeptide whose actions include the stimulation of juvenile hormone biosynthesis, myotropic stimulation, cardioacceleratory functions, and inhibition of active ion transport. Manse-AT is a member of a structurally related peptide family that is widely found in insects and also in other invertebrates. Its precise role depends on the insect species and developmental stage. In some lepidopteran insects including M. sexta, structurally-related AT-like (ATL) peptides can be derived from alternatively spliced mRNAs transcribed from the AT gene. We have isolated a cDNA for an AT receptor (ATR) from M. sexta by a PCR-based approach using the sequence of the ATR from Bombyx mori. The sequence of the M. sexta ATR is similar to several G protein-coupled receptors from other insect species and to the mammalian orexin receptor. We demonstrate that the M. sexta ATR expressed in vertebrate cell lines is activated in a dose-responsive manner by Manse-AT and each Manse-ATL peptide in the rank order ATL-I > ATL-II > ATL-III > AT, and functional analysis in multiple cell lines suggest that the receptor is coupled through elevated levels of Ca(2+) and cAMP. In feeding larvae, Manse-ATR mRNA is present at highest levels in the Malpighian tubules, followed by the midgut, hindgut, testes, and corpora allata, consistent with its action on multiple target tissues. In the adult corpora cardiaca--corpora allata complex, Manse-ATR mRNA is present at relatively low levels in both sexes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Larvae of Galleria mellonella (Greater Wax Moth) have been shown to be susceptible to Campylobacter jejuni infection and our study characterizes this infection model. Following infection with C. jejuni human isolates, bacteria were visible in the haemocoel and gut of challenged larvae, and there was extensive damage to the gut. Bacteria were found in the extracellular and cell-associated fraction in the haemocoel, and it was shown that C. jejuni can survive in insect cells. Finally, we have used the model to screen a further 67 C. jejuni isolates belonging to different MLST types. Isolates belonging to ST257 were the most virulent in the Galleria model, whereas those belonging to ST21 were the least virulent.

Bacterial pathogens either hide from or modulate the host's immune response to ensure their survival. Photorhabdus is a potent insect pathogenic bacterium that uses entomopathogenic nematodes as vectors in a system that represents a useful tool for probing the molecular basis of immunity. During the course of infection, Photorhabdus multiplies rapidly within the insect, producing a range of toxins that inhibit phagocytosis of the invading bacteria and eventually kill the insect host. Photorhabdus bacteria have recently been established as a tool for investigating immune recognition and defense mechanisms in model hosts such as Manduca and Drosophila. Such studies pave the way for investigations of gene interactions between pathogen virulence factors and host immune genes, which ultimately could lead to an understanding of how some Photorhabdus species have made the leap to becoming human pathogens., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Many bacteria persist within phagocytes, deploying complex sets of tightly regulated virulence factors to manipulate and survive within host cells. So far, no single factor has been identified that is sufficient to allow intracellular persistence of an otherwise non-pathogenic bacterium. Here we report that the two-component KdpD/KdpE sensor kinase/response regulator of the insect and human pathogen Photorhabdus asymbiotica (Pa) is sufficient to allow a harmless laboratory strain of E. coli to resist phagocytic killing and persist within insect hemocytes, ultimately killing the insect. Screening of a cosmid library of Pa in E. coli by injection into the moth Manduca sexta, previously identified three overlapping clones which caused the insect to cease feeding and subsequently die. Transposon mutagenesis revealed a cosmid encoded kdp high affinity potassium pump regulon was responsible for this phenotype. Gentamycin protection assays and confocal microscopy revealed the cosmid clones were persisting inside insect hemocytes far longer than control bacteria. Cloning and expression of PakdpD/kdpE alone into E. coli recapitulated the phenotype. Bioassay results and transcriptional analysis of various E. coli kdp mutants harboring the Pa kdp genes confirmed that Pa KdpD/KdpE was able to induce the E. coli kdp pump structural genes in response to exposure to insect hemocytes but not blood plasma alone. The finding that Pa KdpD/KdpE can facilitate resistance of E. coli to phagocytic killing suggests a central role for potassium in this process, supporting previous work implicating potassium sensing in virulence of other bacteria and also in the normal process of protease killing of engulfed bacteria by neutrophils., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Summary: Photorhabdus sp. are entomopathogenic bacteria which, upon experimental infection, interact with the insect immune system, but little is known about the roles of their symbiotic nematode partners Heterorhabditis sp. in natural infections. Here, we investigated the respective contributions of nematodes and bacteria by examining humoral and cellular immune reactions of the model lepidopteran insect Manduca sexta against Heterorhabditis carrying Photorhabdus, nematodes free of bacteria (axenic nematodes) and bacteria alone. Insect mortality was slower following infection with axenic nematodes than when insects were infected with nematodes containing Photorhabdus, or the bacteria alone. Nematodes elicited host immune responses to a lesser extent than bacteria. Transcription of certain recognition and antibacterial genes was lower when insects were naturally infected with nematodes carrying no bacteria compared to insects that received bacteria, either with or without nematodes. Axenic nematodes also did not elicit such high levels of phenoloxidase activity and haemocyte aggregates as did treatments involving Photorhabdus. By contrast, the phagocytic capability of host haemocytes was decreased by both axenic and bacteria-associated nematodes, but not by Photorhabdus alone. These results imply that both bacteria and nematodes contribute separately to the pathogenic modulation of host immune responses during natural infections by the mutualistic Heterorhabdus-Photorhabdus complex.

The degree of plasticization of the alloscutal cuticle of a 'hard' (ixodid) tick, Amblyomma hebraeum, and a 'soft' (argasid) tick, Ornithodoros moubata, was assessed throughout the blood-feeding period. Cuticle viscosity was calculated from rate of creep of cuticle under constant load using a Maxwell model. Feeding-related plasticization (i.e. increased rate of extension under a constant load) occurred in A. hebraeum but not in O. moubata. Maxwell viscosity of unfed A. hebraeum cuticle was relatively high (approximately 720 GPa s) but was significantly lower in feeding ticks. Small partially fed ticks displayed a viscosity of approximately 108 GPa s. Still lower values (42 GPa s) were observed in the largest of the engorged ticks. Following cessation of feeding, there was a significant but limited reversal in viscosity back to approximately 100 GPa s. The water content of cuticle of unfed A. hebraeum (23.4% of wet mass) rose sharply after the onset of feeding and reached a plateau value of 34.0% at a fed/unfed weight ratio of 3 and beyond. Ixodid ticks lay down new endocuticle during the feeding period. The observed increase in cuticle hydration suggests that both old and new cuticles are hydrated during feeding. Monoamines may play an important role in controlling cuticle viscosity. Dopamine (DA) injected into partially fed A. hebraeum caused plasticization. 5-Hydroxytryptamine (serotonin, 5-HT), which induces plasticization in the blood-sucking insect Rhodnius prolixus, had no statistically significant effect on tick cuticle. Octopamine (OA) and tyramine both caused cuticle stiffening (i.e. opposed plasticization). This suggests a possible inhibitory effect but co-injection of OA with DA did not reduce DA-induced plasticization. The mechanism leading to plasticization of tick cuticle may involve a change in cuticular pH. The viscosity of tick cuticle loops was highest at pH 8.0 (389 GPa s) and fell precipitously in the acidic range to a low value of 2.2 GPa s at pH 5.5-5.7. A cuticular pH of approximately 6.5 would account for the lowest viscosity observed under physiological conditions (42.4 GPa s for large, day 0, engorged ticks). The V-ATPase inhibitor, concanamycin A, was a potent inhibitor of DA-induced plasticization. These results are consistent with a model in which DA acts to cause plasticization through transport of H(+) ions into the cuticle. Measurement of cuticular ion (Na(+), K(+), Ca(2+), Mg(2+)) content did not suggest that plasticization is caused by any of these ions. Taken together, our results suggest that the mechanism of cuticular plasticization in feeding A. hebraeum is related to hydration, and involves the transport of H(+) ions into the sub-cuticular space by cells in the hypodermis. Feeding-induced plasticization was not observed in the rapid feeding tick, O. moubata.

The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.

Purpose: To assess primary care providers' communication about breast cancer risk., Methods: We evaluated 86 primary care providers' communication of risk using unannounced standardized (simulated) patients. Physicians were randomly assigned to receive one of three cases: (1) moderate risk case (n = 25), presenting with a breast lump and mother with postmenopausal breast cancer; (2) high-risk (maternal side) case (n = 28), presenting with concern about breast cancer risk; and (3) high-risk (paternal side) case (n = 33), presenting with an unrelated problem. After the appointment, three qualitative parameters were assessed by standardized patients on a 3-point scale (3 = highest satisfaction, 1 = lowest): whether the physician took adequate time; acknowledged her concerns; and offered reassurance., Results: Mean satisfaction with physician communication was higher for the moderate risk case (2.92) than for the high-risk paternal case (2.25) or high-risk maternal case (2.42) (P < 0.0001). The score was not influenced by session length, medical specialty, or physician gender., Conclusion: Physicians more consistently provided a moderate risk standardized patients with reassurance and support compared with the high-risk cases. Primary care physicians may be more unprepared or uneasy addressing the issues raised by more complex scenarios and may benefit from training in the assessment and communication of breast cancer risk.

Injecting the insect pathogenic bacterium Photorhabdus luminescens into the blood system of the model lepidopteran insect Manduca sexta induces nitric oxide synthase (NOS) expression in the fat body and blood cells (haemocytes), whereas following oral ingestion of bacteria NOS expression is limited to the gut. We used RNA interference to knock-down expression of NOS throughout the insect. Preventing NOS induction in this way adversely affected the survival of orally infected insects and caused a significant increase in the number of bacteria crossing into the haemolymph. By contrast, knock-down of NOS had no effect on the mortality rate of insects infected with P. luminescens by injection. Pharmacological inhibition of NOS decreased both nitric oxide (NO) levels in the gut wall and survival of orally infected insects, whereas elevation of gut wall NO using an NO donor increased survival of NOS silenced caterpillars. Together, our results imply that induced synthesis of NO is important in mediating insect immune defence against the pathogen by inhibiting transfer of bacteria across the gut wall.

Insect hemocytes (blood cells) are a central part of the insect's cellular response to bacterial pathogens, and these specialist cells can both recognize and engulf bacteria. During this process, hemocytes undergo poorly characterized changes in adhesiveness. Previously, a peptide termed plasmatocyte-spreading peptide (PSP), which induces the adhesion and spreading of plasmatocytes on foreign surfaces, has been identified in lepidopteran insects. Here, we investigate the function of this peptide in the moth Manduca sexta using RNA interference (RNAi) to prevent expression of the precursor protein proPSP. We show that infection with the insect-specific bacterial pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli induces proPSP mRNA transcription in the insect fat body but not in hemocytes; subsequently, proPSP protein can be detected in cell-free hemolymph. We used RNAi to silence this upregulation of proPSP and found that the knock-down insects succumbed faster to infection with P. luminescens, but not E. coli. RNAi-treated insects infected with E. coli showed a reduction in the number of circulating hemocytes and higher bacterial growth in hemolymph as well as a reduction in overall cellular immune function compared with infected controls. Interestingly, RNAi-mediated depletion of proPSP adversely affected the formation of melanotic nodules but had no additional effect on other cellular responses when insects were infected with P. luminescens, indicating that this pathogen employs mechanisms that suppress key cellular immune functions in M. sexta. Our results provide evidence for the central role of PSP in M. sexta cellular defenses against bacterial infections.

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo. Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli. The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.

Family history is increasingly important in primary care as a means to detect candidates for genetic testing or tailored prevention programs. We evaluated primary care physicians' skills in assessing family history for breast cancer risk, using unannounced standardized patient (SP) visits to 86 general internists and family medicine practitioners in King County, WA. Transcripts of clinical encounters were coded to determine ascertainment of family history, risk assessment, and clinical follow-up. Physicians in our study collected sufficient family history to assess breast cancer risk in 48% of encounters with an anxious patient at moderate risk, 100% of encounters with a patient who had a strong maternal family history of breast cancer, and 45% of encounters with a patient who had a strong paternal family history of breast and ovarian cancer. Increased risk was usually communicated in terms of recommendations for preventive action. Few physicians referred patients to genetic counseling, few associated ovarian cancer with breast cancer risk, and some incorrectly discounted paternal family history of breast cancer. We conclude that pedigree assessment of breast cancer risk is feasible in primary care, but may occur consistently only when a strong maternal family history is present. Primary care education should focus on the link between inherited breast and ovarian cancer risk and on the significance of paternal family history. Educational efforts may be most successful when they emphasize the value of genetic counseling for individuals at risk for inherited cancer and the connection between genetic risk and specific prevention measures., (2009 Wiley-Liss, Inc.)

Current sequence databases now contain numerous whole genome sequences of pathogenic bacteria. However, many of the predicted genes lack any functional annotation. We describe an assumption-free approach, Rapid Virulence Annotation (RVA), for the high-throughput parallel screening of genomic libraries against four different taxa: insects, nematodes, amoeba, and mammalian macrophages. These hosts represent different aspects of both the vertebrate and invertebrate immune system. Here, we apply RVA to the emerging human pathogen Photorhabdus asymbiotica using "gain of toxicity" assays of recombinant Escherichia coli clones. We describe a wealth of potential virulence loci and attribute biological function to several putative genomic islands, which may then be further characterized using conventional molecular techniques. The application of RVA to other pathogen genomes promises to ascribe biological function to otherwise uncharacterized virulence genes.

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.

Photorhabdus bacteria live in a 'symbiosis of pathogens' with nematodes that invade and kill insects. Recent work has begun to use the power of the model insect Drosophila to dissect the molecular basis of the invertebrate immune response to the combined insult of the worms and their symbiotic bacterial pathogens. By using RNA interference, it is now also possible to dissect this complex tripartite interaction in a range of both model and non-model hosts.

Bacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knock-down of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.

Photorhabdus is a virulent pathogen that kills its insect host by overcoming immune responses. The bacterium also secretes a range of antibiotics to suppress the growth of other invading microorganisms. Here we show that Photorhabdus produces a small-molecule antibiotic (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene (ST) that also acts as an inhibitor of phenoloxidase (PO) in the insect host Manduca sexta. The Photorhabdus gene stlA encodes an enzyme that produces cinnamic acid, a key precursor for production of ST, and a mutation in stlA results in loss of ST production and PO inhibitory activity, which are both restored by genetic complementation of the mutant and also by supplying cinnamic acid. ST is produced both in vitro and in vivo in sufficient quantities to account for PO inhibition and is the only detectable solvent-extractable inhibitor. A Photorhabdus stlA- mutant is significantly less virulent, proliferates slower within the host, and provokes the formation of significantly more melanotic nodules than wild-type bacteria. Virulence of the stlA- mutant is also rescued by supplying cinnamic acid. The proximate cause of the virulence effect, however, is the inhibition of PO, because the effect of the stlA- mutation on virulence is abolished in insects in which PO has been knocked down by RNA interference (RNAi). Thus, ST has a dual function both as a PO inhibitor to counter host immune reactions and also as an antibiotic to exclude microbial competitors from the insect cadaver.

Prior infection of Manduca sexta caterpillars with the non-pathogenic bacterium Escherichia coli elicits effective immunity against subsequent infection by the usually lethal and highly virulent insect pathogen Photorhabdus luminescens TT01. Induction of this protective effect is associated with up-regulation of both microbial pattern recognition protein genes (hemolin, immulectin-2 and peptidoglycan recognition protein) and anti-bacterial effector genes (attacin, cecropin, lebocin, lysozyme and moricin). We used RNA interference to knock down over-transcription of members of both these sets of genes one at a time. Interfering with expression of individual recognition proteins had a drastic adverse effect on the E. coli elicited immunity. RNAi knock-down of immulectin-2 caused the greatest reduction in immunity, followed by hemolin and peptidoglycan recognition protein (PGRP) in that order, to the extent that knock-down of any one of these three proteins left the insects more susceptible to P. luminescens infection than insects that had not experienced prior infection with E. coli. Interfering with the expression of individual antibacterial effector proteins and peptides had a much less marked effect on immunity. Knock-down of attacin, cecropin or moricin caused treated insects to be more susceptible to P. luminescens infection than controls that had been pre-infected with E. coli but which had not received the specific RNAi reagents, but they were still less susceptible than insects that had not been pre-infected with E. coli. RNAi knock-down with expression of lebocin or lysozyme had no effect on E. coli-induced immunity to P. luminescens, indicating that these effectors are not involved in the response. By bleeding pre-infected caterpillars and growing the pathogen directly within cell-free insect haemolymph, we showed that at least part of the protection elicited by previous exposure to E. coli is due to the presence of factors within the blood plasma that inhibit the growth of P. luminescens. The production of these factors is inhibited by RNAi treatment with ds-RNA reagents that knock down hemolin, immulectin-2, and PGRP. These results demonstrate that the insect immune system can be effectively primed by prior infection with non-pathogenic bacteria against subsequent infection by a highly virulent pathogen. Given the continuous normal exposure of insects to environmental and symbiotic bacteria, we suggest that prior infection is likely to play a significant and underestimated role in determining the level of insect immunity found in nature.

Bacterial pathogens either hide from or overcome the immune response of their hosts. Here we show that two different species of insect pathogenic bacteria, Photorhabdus luminescens TT01 and Photorhabdus asymbiotica ATCC43949, were both recognized by the immune system of their host Manduca sexta, as indicated by a rapid increase in the levels of mRNAs encoding three different inducible microbial recognition proteins, Hemolin, Immulectin-2 and peptidoglycan recognition protein. RNA interference (RNAi)-mediated inhibition of expression ("knock-down") of each of these genes at the level of both mRNA and protein was achieved through injection of double-stranded RNA (dsRNA). Knock-down of any one of these genes markedly decreased the ability of the insects to withstand infection when exposed to either species of Photorhabdus, as measured by the rate at which infected insects died. RNAi against Immulectin-2 caused the greatest reduction in host resistance to infection. The decreased resistance to infection was associated with reduced hemolymph phenoloxidase activity. These results show not only that Photorhabdus is recognized by the Manduca sexta immune system but also that the insect's immune system plays an active, but ultimately ineffective, role in countering infection.

We have discovered a new type of haemocyte in the larval stage of the tobacco hornworm moth Manduca sexta that has extreme phagocytic ability; each cell can engulf up to 500 bacteria. This level of phagocytosis may be unprecedented among animal cells. Although these hyperphagocytic cells (HP) only represent about 1% of the circulating haemocytes, they are responsible for sequestering the majority of the bacteria by circulating haemocytes when non-pathogenic, heat-killed Escherichia coli are injected into the haemolymph. Extreme phagocytosis by HP is not limited to Gram-negative bacteria since heat-killed Staphylococcus aureus as well as positively and negatively charged microspheres are also highly phagocytosed. Evidence is presented to show that phagocytosis by HP is involved in the early stages of nodule formation in infected insects. In addition, HP are also present in non-infected insects, characterised by their distinctive spreading morphology, which becomes impaired following hyperphagocytosis of bacteria. This is the first time that a dedicated "professional" phagocytic class of haemocyte has been reported for an invertebrate. The importance of these specialised cell types in the M. sexta immune response and their role in nodule formation is discussed.

Fungus garden material from recently established Atta sexdens rubropilosa colonies (6-12 months old) was sampled to detect antibiotic producing microorganisms that inhibited the growth of pathogens of insects and of the fungus gardens but did not affect their mutualistic fungus. A bacterium with activity against the entomopathogenic fungus Beauveria bassiana was isolated from 56% of the gardens tested (n=57) and identified from its biochemical profile and from 16S and 23S ribosomal DNA sequences as a member of the genus Burkholderia. The ant-associated Burkholderia isolates secreted a potent, anti-fungal agent that inhibited germination of conidia of the entomopathogenic fungi B. bassiana, Metarhizium anisopliae, of the saprophytic Verticillium lecanii, and also of a specialist fungus garden Escovopsis weberi. Growth of the ant's mutualist fungus was unaffected.

The ability to adhere to and spread on a surface is a common property of insect blood cells. Spreading on a glass surface by insect hemocytes is often used as a measure of immune fitness that can be inhibited by some insect pathogens and parasites. Here, we report that upon infection of the tobacco hornworm Manduca sexta with either a fungus (Beauveria bassiana) or a bacterium (Photorhabdus luminescens), a new type of hemocyte, not previously observed in healthy insects, was found in hemocyte monolayers. These cells have a distinctive morphology, characterised by extreme spreading ability. They achieve a diameter of up to 120 microm after 1 h on glass coverslips and are therefore extremely thin. These hyper-spreading cells first appear in fungal-infected insects prior to hyphal growth. Their numbers later fall to zero as the pathogen begins to proliferate. The same hyper-spreading cells are induced after a 24 h delay following an injection of laminarin, a source of the fungal cell wall polymer beta-1,3-glucans. Wounding, on the other hand, did not cause the appearance of hyper-spreading cells. Evidence is presented here that is consistent with these spreading cells having a role in the cellular immune response of nodule formation.

Sublethal concentrations of the bisacylhydrazine moulting hormone agonists, RH-5849, and tebufenozide (RH-5992) were fed to sixth (final) instar larvae of Spodoptera litura. Both RH-5849 and tebufenozide adversely affected the mating success of S. litura when the surviving treated males were crossed with normal females. The ecdysone agonists decreased the longevity of treated males and of untreated females when crossed with treated males. The number of eggs laid by untreated females mated to treated males was decreased, and the fertility (percentage of hatching success) of the resulting eggs was reduced. These effects on male reproductive success were at least in part explained by a reduction in the number of sperm transferred during mating. The adverse effects of tebufenozide on male reproductive function were qualitatively the same as those of RH-5849, but tebufenozide was active at lower concentrations. To understand the reason for these adverse effects on male reproduction, we investigated the effects of the insecticides on male reproductive physiology. Male reproductive tract development and testicular volume of resulting adult moths were adversely affected by sublethal larval exposure to the ecdysone agonists. Dose-dependent reductions occurred in the production of eupyrene and apyrene spermatozoa in the adult testes, and in the number of spermatozoa released from the testes into the male reproductive tract. The descent into the male tract of both eupyrene and apyrene sperm was found to start at the normal stage of development in both normal and treated insects, but the daily rhythm of sperm descent was subsequently disturbed in the insecticide-treated moths. This affected the numbers of sperm in the upper vas deferens (UVD), seminal vesicle (SV), and duplex (duplex). Injections of RH-5849 given to pharate adult or newly emerged adult S. litura also caused drastic reduction in the number of sperm in the upper regions of the male tract, when measured 24 h after injection. The possible importance of pest population reduction through the sublethal anti-reproductive effects of insecticides is discussed.

Photorhabdus are insect pathogenic bacteria that replicate within the insect haemocoel following release from their entomopathogenic nematode symbionts. To investigate how they escape the cellular immune response we examined the effects of two strains of Photorhabdus, W14 and K122, on Manduca sexta phagocytes (haemocytes), in vitro and in vivo. Following injection of Esherichia coli into Manduca larvae, these non-pathogenic bacteria are rapidly cleared from the haemolymph and the number of free haemocytes transiently increases. In contrast, following injection of either strain of pathogenic Photorhabdus, the bacteria grow rapidly while the number of haemocytes decreases dramatically. In vitro incubation of haemocytes with either Photorhabdus supernatant reduced haemocyte viability, and the W14 supernatant caused distinct changes in the actin cytoskeleton morphology of different haemocyte cell types. In phagocytosis assays both Photorhabdus strains can inhibit their own phagocytosis whether the bacterial cells are alive or dead. Further, the supernatant of W14 also contains a factor capable of inhibiting the phagocytosis of labelled E. coli. Together these results suggest that Photorhabdus evades the cellular immune response by killing haemocytes and suppressing phagocytosis by mechanisms that differ between strains.

Invertebrates, including insects, are being developed as model systems for the study of bacterial virulence. However, we understand little of the interaction between bacteria and specific invertebrate tissues or the immune system. To establish an infection model for Photorhabdus, which is released directly into the insect blood system by its nematode symbiont, we document the number and location of recoverable bacteria found during infection of Manduca sexta. After injection into the insect larva, P. luminescens multiplies in both the midgut and haemolymph, only later colonizing the fat body and the remaining tissues of the cadaver. Bacteria persist by suppressing haemocyte-mediated phagocytosis and culture supernatants grown in vitro, as well as plasma from infected insects, suppress phagocytosis of P. luminescens. Using GFP-labelled bacteria, we show that colonization of the gut begins at the anterior of the midgut and proceeds posteriorly. Within the midgut, P. luminescens occupies a specific niche between the extracellular matrix and basal membrane (lamina) of the folded midgut epithelium. Here, the bacteria express the gut-active Toxin complex A (Tca) and an RTX-like metalloprotease PrtA. This close association of the bacteria with the gut, and the production of toxins and protease, triggers a massive programmed cell death of the midgut epithelium.

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect host's immune system., ((c) 2002 Elsevier Science (USA).)

Sperm production and movement from the fused testes into the male reproductive tract of the common cutworm Spodoptera litura were studied in insects maintained in a 12h:12h light dark (LD) regime. Two types of sperm bundles, eupyrene (nucleated) and apyrene (anucleate) were present in the adult testes. Eupyrene bundles constituted about 25% of the total. Descent of spermatozoa from the testes into the upper vas deferens (UVD) first occurred about 24-30h before adult eclosion. On entering the reproductive tract, eupyrene spermatozoa remained in bundles while apyrene bundles became dissociated before they reached the UVD. Downward movement of both eupyrene and apyrene spermatozoa within the male tract occurred in a daily rhythm. Sperm descent from the testes into the UVD occurred during the early scotophase, followed by their further descent into the seminal vesicle (SV) during the photophase. Spermatozoa remained in the SV for only a short duration, whence sperm quickly passed through the lower vas deferens into the duplex, which acted as the main sperm storage organ until mating was initiated. During mating 80% of sperm left the duplex, but mating did not influence the number of sperm bundles that subsequently descended into the duplex or the rate of their descent. There was no evidence of sperm reflux. Rearing in constant light (LL) and in constant dark (DD) reduced the number of eupyrene sperm present in the testes of adults that emerged in LL and DD compared to controls (LD), although there was no significant effect on the number of apyrene sperm in the testes. The rhythmic pattern of sperm descent was suppressed in both LL and DD regimes, and the number of sperm in the duplex was adversely affected, with a marked impact in LL reared insects. Male longevity, mating behaviour, oviposition and fertility were found to be more severely affected in LL than in DD.

Nicotinic acetylcholine receptors (nAChR) of insect and other invertebrates are heterogeneous and new tools are needed to dissect their multiplicity. [(3)H]-Methyllycaconitine ([(3)H]-MLA) is a novel radioligand which is a potent antagonist at vertebrate alpha7-type nAChR. Putative invertebrate nAChR of the aphid Myzus persicae, the moths Heliothis virescens and Manduca sexta, the fly Lucilia sericata, and the squid Loligo vulgaris were investigated in radioligand binding studies with [(3)H]-MLA. Saturable binding was consistent with a single class of high affinity binding sites for each of these invertebrates, characterised by a dissociation constant, K(d), of approximately 1 nM and maximal binding capacities, B(max), between 749 and 1689 fmol/mg protein for the insects and 14,111 fmol/mg protein for squid. [(3)H]-MLA binding to M. persicae membranes was characterised in more detail. Kinetic analysis demonstrated rapid association in a biphasic manner and slow, monophasic dissociation. Displacement studies demonstrate the nicotinic character of [(3)H]-MLA binding sites. Data for all nicotinic ligands, except MLA itself, are consistent with displacement from a high and a low affinity site, indicating that displacement is occurring from two or more classes of nicotinic binding site that are not distinguished by MLA itself. Autoradiographic analysis of the distribution of [(3)H]-MLA binding sites in Manduca sexta shows discrete labelling of neuropil areas of the optic and antennal lobes.

Context: Women commonly misunderstand their risk for breast cancer, overestimating both their risk for developing the disease at a young age and their lifetime risk., Objective: To determine whether age bias occurs in popular media coverage of breast cancer. SELECTION STRATEGY: The search term breast cancer was used to identify 389 articles in U.S. magazines with a circulation of at least 500,000 published between January 1, 1993, and June 30, 1997., Main Outcome Measures: Presence of age-related themes and age of patients with breast cancer who were described in vignettes., Results: Age-related themes included breast cancer as a cause of premature death, breast cancer in mothers of young children, and the impact of a breast cancer diagnosis on dating and marriage. Factual information about age as a risk factor for breast cancer was presented in only 14% of articles, and age was often included in vignettes describing a woman with breast cancer. Thirty-four percent of the articles included one or more breast cancer vignettes. These articles included 172 unique vignettes in which patient age was described. In 84% of the vignettes (144 of 172), women were diagnosed with breast cancer before 50 years of age; in 47% (80 of 172), women were diagnosed before 40 years of age. On the basis of the age-specific incidence of breast cancer in the United States, the expected percentages would be 16% and 3.6%, respectively., Conclusions: Stories about breast cancer in popular U.S. magazines misrepresent the age distribution of the disease, emphasizing atypical cases of early-onset breast cancer and their social consequences. This presentation of breast cancer may contribute to women's fears of breast cancer and to overestimates of personal risk.